WO2000050448A1 - A method for controlling an enzymatic reaction by the alpha and beta domains of metallothionein - Google Patents

A method for controlling an enzymatic reaction by the alpha and beta domains of metallothionein Download PDF

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WO2000050448A1
WO2000050448A1 PCT/US2000/004734 US0004734W WO0050448A1 WO 2000050448 A1 WO2000050448 A1 WO 2000050448A1 US 0004734 W US0004734 W US 0004734W WO 0050448 A1 WO0050448 A1 WO 0050448A1
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domain
metallothionein
alpha
beta
domains
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PCT/US2000/004734
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French (fr)
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Bert L. Vallee
Wolfgang Maret
Edmond H. Fischer
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Vallee Bert L
Wolfgang Maret
Fischer Edmond H
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Priority to AU30066/00A priority Critical patent/AU3006600A/en
Publication of WO2000050448A1 publication Critical patent/WO2000050448A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/825—Metallothioneins

Definitions

  • the present invention relates to the alpha and beta domains of metallothionein and analogs thereof, their synthesis, and methods for controlling in vitro and industrial enzymatic reactions using them.
  • Purified metal-free and metal-containing alpha and beta domains of metallothionein and analogs thereof are provided as described below.
  • a high yield method of synthesis and purification is also provided for the metal-free
  • Metallothionein was discovered in 1957 (Margoshes, M. and Nallee, B. L., 1957, J. Am. Chem. Soc. 79, 4813). By all counts it is a most unusual and
  • MT usually binds seven zinc atoms, but it can also contain copper, cadmium, iron and traces of other metals. In an evolutionary sense it is a very old protein.
  • MT-1 and MT-2 are the two prevalent forms, which are expressed, but whose physiological functions are unknown.
  • MT-3 was discovered only recently in human brains
  • the number of genes that code for human MTs could be as high as 17.
  • T avidly binds metal ions and is highly susceptible to proteolytic digestion and oxidation. T is a very efficient biological chelating agent for zinc. It suppresses the D ⁇ A-binding capacity of zinc finger transcription factors in vitro by sequestering zinc and removing it from their structural sites (Zeng, J., Heuchel, R., Schaffher, W.
  • agents such as glutathione or citrate, which can bind zinc themselves, but do not remove it from the active site of the zinc metalloenzyme, can serve in the transfer process (Jacob, C, Maret, W. and Vallee, B. L., 1998, Proc. ⁇ atl. Acad. Sci. USA 95, 3489-3494).
  • zinc In addition to its catalytic role in more than 300 zinc metalloenzymes and its structural role in an even greater number of non-enzymatic proteins, zinc also is a known inhibitor of enzymes in general, including zinc metalloenzymes.
  • zinc binding experiments demonstrated a second, non-active zinc site in carboxypeptidase A (Coleman, J. E. and Vallee, B. L., 1960, J. Biol. Chem. 235, 390-399). Twenty years later this was established as an inhibitory site which was later on shown to be located in the vicinity (3.3 A) of the catalytic zinc atom and to inhibit the enzyme with an apparent K ] value of 24 ⁇ M (Larsen, K. L. and Auld, D.
  • the K, values for zinc inhibition of bacterial adenylosuccinate synthase and 6-phosphogluconate dehydrogenase are 29 and 21 nM, respectively (Kang, C. and Fromm, H. J., 1995, J. Biol. Chem. 270, 15539-15544; Niehaus, W. G., Richardson, S. B. and Wolz, R. L., 1996, Arch. Biochem. Biophys. 333, 333-337). Their eukaryotic counterparts, however, have not been examined as yet in this regard.
  • Protein Phosphorylation and Dephosphorylation plays a central role in metabolism and cellular signaling.
  • Protein kinases (PK) and protein phosphorylases (PP) play complimentary roles in signal transduction from the extracellular environment to the cytosol.
  • PK Protein kinases
  • PP protein phosphorylases
  • insulin receptor transduction involves tyrosine kinase domain activation on binding of insulin, with subsequent phosphorylation of target cytosol proteins by the activated receptor.
  • Protein phosphatases remove inorganic phosphate from phosphorylated proteins, and thus attenuate signals involving phosphorylated proteins.
  • PTP Protein-tyrosine phosphatases
  • PSP protein serine/threonine phosphatases
  • at least one oncogene is known to code for a protein-tyrosine kinase (PTK).
  • Intrinsic PTP activity is known to be two to three orders of magnitude greater than PTK activity, and thus the cellular levels of endogenous phosphorylated tyrosine residues is ordinarily low. All PTPs share a common signature motif from bacterial to mammalian enzymes.
  • Iron, copper, and zinc are known to be essential to most or all forms of life. Additionally, vanadium, chromium, manganese, cobalt, nickel, arsenic, selenium, molybdenum and tungsten are known to be essential to some or many life forms. Many of these metals function as part of metalloproteins, either structurally, or in the case of some metalloenzymes, catalytically. Furthermore, metals such as lithium are known to have pharmacological effects.
  • Zinc in MT is bound extremely tightly (K D about 10 "13 M) (Kagi, J. H. R., 1993, in Metallothionein III, eds. Suzuki, K. T., Imura, N. and Kimura, M. (Birkhauser, Basel), p. 29).
  • MT metallothionein
  • GSH glutathione
  • T thionein
  • EDTA ethylenediaminetetraacetic acid
  • finoc 9-fluorenylmethyloxycarbonyl
  • t-boc or boc tertiary-butyloxycarbonyl
  • the term compound means any molecule, salt, metal, or any other combination of one or more atoms, including but not limited to covalently bonded molecules, ionic materials, metallic materials, crystalline materials, atoms or molecules or ions in solution, atoms or molecules or ions in the gas phases, and combinations of any of the preceding.
  • metallothionein includes all of the isoforms of metallothionein- 1, metallothionein-2, metallothionein-3, and metallothionein-4; furthermore the primary sequence of such metallothionein can correspond to that of any species known to produce metallothionein.
  • domain means a synthetic polypeptide synthesized by the procedures of the present invention, that (1) has the same primary structure as the alpha domain of metallothionein, (2) has the same primary structure as the beta domain of metallothionein, or (3) is an analogous polypeptide (hereinafter "analog") having a closely related primary structure to either the alpha or beta domain of metallothionein which analogous polypeptide is capable of binding metals in a cluster arrangement similar to the arrangements of the alpha or beta domain of metallothionein.
  • analogous polypeptide hereinafter “analog”
  • domain also includes derivatives of domains. Unless otherwise specified, the term domain shall include metal-free and metal-containing domains, and for purposes of the present invention, selenium shall be considered to be included in the term metal.
  • the term derivative means a molecule (including but not 0 limited to amino acids and peptides) chemically modified, for example by binding to that molecule another atom, molecule or ion, where such other molecule or ion is either monomeric or polymeric.
  • sequence means two or more amino acids, and where there is more than one amino acid, the amino acids are covalently bonded through 5 one or more peptide bonds, with the number of peptide bonds being one less than the number of amino acids.
  • FIG. 1 Inhibition of caspase-3 by zinc. Caspase-3, 1.7 nM, was incubated with 30 various amounts of zinc. Activity was assayed after 5 min.
  • FIG. 3 Reactivation of zinc-inhibited glyceraldehyde 3-phosphate dehydrogenase with T.
  • Glyceraldehyde 3-phosphate dehydrogenase 10 nM
  • ZnSO 4 1 ⁇ M
  • Enzyme activity was measured for 0.5 min, T, 250 nM, was then injected into the cuvet, and the measurement continued.
  • FIG. 7 Reconstitution of zinc-depleted sorbitol dehydrogenase (apo-SDH) with the clusters.
  • the beta (A) and alpha (B) domain were incubated with apo-SDH (1.7 ⁇ M) at different ratios between zinc and apo-SDH.
  • the present invention provides a method of synthesis and purification of a domain having a primary amino acid sequence comprising the following steps:
  • alpha amino acids are selected from the group consisting of amino acids with aliphatic group containing side chains wherein said aliphatic group is hydrogen or alkyl, amino acids with aromatic group containing side chains, amino acids with sulfur group containing side chains wherein said sulfur group is a thiol or a thioether, amino acids with hydroxyl group containing side chains, amino acids with amine group containing side chains, amino acids with guanidinium group containing side chains, amino acids with carboxylate group containing side chains, and amino acids with amide group containing side chains, wherein the alpha amino groups are protected with a protecting group selected from the group consisting of Fmoc, t-Boc, and CBZ, the carboxylate groups are protected with a protecting group selected from the group consisting of t-butyl ester and benzyl ester, the hydroxyl groups are protected with a protecting group
  • step (c) purifying the peptide obtained from step (b);
  • step (d) precipitating the peptide obtained from step (c);
  • substitution of one or more cysteine residues with selenocysteine substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with
  • one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine,
  • one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine
  • the method step (a) is accomplished using an automated solid- phase synthesizer.
  • the domain is the alpha domain of metallothionein or the beta domain of metallothionein.
  • the alpha amino groups are protected with an Fmoc protecting group
  • the carboxylate groups are protected with a t- butyl ester protecting group
  • the hydroxyl groups are protected with a t-butyl ether protecting group
  • the amine groups are protected with a t-Boc protecting group.
  • the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; and the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid.
  • the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.
  • the domain synthesized according to the method is the alpha domain of metallothionein or the beta domain of metallothionein; and wherein the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t-butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group; wherein the synthesizing step (a) is accomplished using a solid-phase synthesizer wherein the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; wherein the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydr
  • all the alpha amino groups are protected with an Fmoc protecting group
  • all the side chain carboxylic acid groups are protected with a t-butyl ester protecting group
  • all side chain hydroxyl groups are protected with a t-butyl ether protecting group
  • all side chain amine groups are protected with a t-Boc protecting group.
  • the cleavage of the peptide from the solid support and removal of the non- acetimidomethyl protecting groups is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid.
  • the purification by gel filtration chromatography is accomplished using a gel prepared from Sephadex-25 which comprises beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and preferably, the Sephadex gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid. Also, preferably, the removal of the acetimidomethyl protecting group is accomplished with a solution comprising silver(I) nitrate in acetic acid.
  • the invention also provides the domains according to method of synthesis and purification. These domains may be metal containing or metal- free. Where metal containing, the metal may be selected from the group consisting of main group metals, transition metals, lanthanides, and actinides. Preferred metals of the invention are zinc, copper, gold, cadmium, iron, cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technetium, lanthanum, and combinations thereof. Most preferably, the metal is zinc.
  • compositions comprising a domain, wherein said domain comprises no more than 5 percent impurities arising from the synthesis and isolation of the domain, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
  • one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine,
  • the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of (i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein,
  • the domain of this composition comprises no more than 2 percent impurities arising from the synthesis and isolation of the domain.
  • the invention further provides a composition comprising a domain, wherein said domain comprises at least 95 % of the total protein in said composition, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
  • one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii); and wherein the primary
  • the domain of this composition comprises at least 98 % of the total protein in said composition.
  • compositions comprising a domain, wherein said domain comprises no more than 5 percent impurities arising from the synthesis and isolation of the domain, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of (i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (i ⁇ ) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cyst
  • one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and
  • the domain of this composition comprises no more than 2 percent impurities arising from the synthesis and isolation of the domain.
  • the invention further provides a composition comprising a domain, wherein said domain comprises at least 95 % of the total protein in said composition, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
  • substitution of one or more cysteine residues with selenocysteine substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with
  • one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine,
  • one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine
  • the domain of this composition comprises at least 98 % of the total protein in said composition.
  • a domain of the present invention includes one or more probe characteristics.
  • probe characteristics useful in the invention are incorporation of one or more radioactive metals into a domain, isotopic substitution of one or more atoms in the domain, fusion to a domain of one or more of a fluorophore, an NMR contrast reagent, or a Foerster radiation-less energy transfer reagent, and arsenoazo modification of amino acid residues such as tyrosine.
  • the domains may be formulated as pharmaceutical compositions.
  • Such pharmaceutical compositions comprise one or more domains and further comprise one or more ingredients selected from the group consisting of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a wetting agent, a buffering agent, an emulsifying agent, and a binding agent.
  • These compositions may also use domains wherein the domains are immobilized on a biologically-inert non-polymeric or polymeric support.
  • This support may comprise, but is not limited to, a material selected from the group consisting of a polymeric support in a form selected from a bead, a fiber, and a sheet; paper; and cotton thread.
  • the polymeric support comprises a functionalized polystyrene.
  • the invention provides methods for controlling enzymatic reactions susceptible to such control, the reactions comprising conversion of a substrate using the enzyme to a product; the method comprising adding an amount of a composition comprising a domain to a reaction mixture comprising an enzyme; wherein the amount is sufficient to effect a change in a rate of the reaction, wherein the change is selected from the group consisting of an increase in the rate of reaction by a factor of at least about 1.1 and a decrease in rate of reaction by a factor of at least about 1.1; wherein the enzyme comprises an inhibitory site that binds a metal cation; wherein the domain is added to the reaction mixture at a time selected from the group consisting of a time before which the substrate is available to the enzyme and a time during which the substrate is available to the enzyme; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metall
  • substitution of one or more cysteine residues with selenocysteine substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with
  • one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine,
  • one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine
  • the domain is the alpha domain of metallothionein or the beta domain of metallothionein.
  • the domain is an analog of the alpha domain of metallothionein wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of (ii) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, ( ⁇ ) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by
  • the domain is an analog of the alpha domain of metallothionein whose primary amino acid sequence differs from the primary amino acid sequence of the alpha domain of metallothionein by fusion of one primary sequence of the beta domain of metallothionein to the N-terminal end of the alpha domain of metallothionein.
  • the domains useful in controlling enzymatic reactions may be purified. They may comprise no more than 5 percent impurities arising from the synthesis and isolation of the domain, and more preferably, no more than 2 percent impurities arising from the synthesis and isolation of the domain.
  • the domains comprise at least 95 % of the total protein in the composition, and preferably, at least 98 % of the total protein in the composition.
  • the domains may be metal free or metal containing, and the domains may contain one or more metals selected from the group consisting of main group metals, transition metals, lanthanides, and actinides.
  • the metal containing domains will contain one or more metals selected from the group consisting of zinc, copper, gold, cadmium, iron, cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technetium, lanthanum, and combinations thereof, and most preferably, will contain zinc.
  • metals selected from the group consisting of zinc, copper, gold, cadmium, iron, cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technetium, lanthanum, and combinations thereof, and most preferably, will contain zinc.
  • the enzymes of the reactions that may be controlled are preferably selected from the group consisting of glucose isomerases, amylolytic enzymes, proteases, lipases, pectases, and xylose isomerases.
  • the domain preferably contains at least one metal selected from the group consisting of Ag(I), Ni(II), Hg(II), Fe(II), Zn(II), Ca(II),Al(III), Ga(II), Ba(II), As(III), and Cu(I, II) and combinations of these metals.
  • the domain is preferably metal-free.
  • the enzymatic reactions controlled by the domains of the present invention are also preferably used in enzyme-based cleaning agents, where the cleaning agent is preferably selected from the group consisting of enzyme-based laundry detergents, dishwashing detergents, grease removers, and spot removers. Also preferred are enzymatic reactions used in assays, and these assays are preferably clinical assays or industrial assays.
  • the method of the present invention is useful in controlling enzymatic reactions used to make products that are organic molecules selected from the group consisting of an intermediate in an organic synthesis and a final product of an organic synthesis. Also, the method of the present invention is useful in controlling enzymatic reactions used to make a biomolecule selected from the group consisting of an intermediate in a biochemical process and a final product of a biochemical process.
  • the present invention relates to the alpha and beta domains of metallothionein and analogs thereof, their synthesis, therapeutic applications, and methods for controlling in vitro and industrial enzymatic reactions using them.
  • Purified metal-free and metal-containing alpha and beta domains of metallothionein and analogs thereof are provided as described below.
  • a high yield method of synthesis and purification is also provided for the metal-free and metal containing alpha and beta domains of metallothionein and analogs thereof.
  • methods for controlling in vitro and industrial enzymatic reactions are provided wherein the enzymatic processes are controlled by transfer of zinc to an enzyme zinc-inhibitory site or removal of zinc from an enzyme zinc inhibitory site using the alpha and beta domains of metallothionein and analogs thereof.
  • polypeptides synthesized by the procedures described below include purified synthetic polypeptides with the same primary structure (i.e. amino acid sequence) as the alpha or beta domains of metallothionein, where these polypeptides have been synthesized according to the synthetic procedures described below.
  • sequences of the polypeptides correspond to the sequences of the alpha and beta domains of the isoforms of human metallothionein, including the four known isoforms (MT-1 through 4) and those as yet unidentified. More preferably, the sequences of the polypeptides correspond to the sequences of the alpha and beta domains of human metallothionein-2 (MT-2).
  • the primary sequence of human metallothionein-2 beta domain is:
  • the primary sequence of human metallothionein-2 alpha domain is:
  • the present invention is also directed to analogs of the alpha or beta domains of metallothionein, where these analogs are also synthesized according to the synthetic procedures described below.
  • the primary sequences of these analogs are related to but not identical to those of the alpha or beta domains of metallothionein.
  • the tertiary structures of these analogs maintain similar three-dimensional arrangements of the cysteine residues to those in the alpha or beta domains of metallothionein.
  • these analogs are capable of binding metal atoms in an analogous fashion to the metal binding of the alpha or beta domains of metallothionein.
  • the change in primary structure will accomplish one or more of the following desired effects: (1) it will change the metal binding constants so as to facilitate metal release or so as to inhibit release of metals picked up through administration of the metal- free domains; (2) it will provide suitable functionality for derivatizing and/or solubilizing the domains; and (3) it will provide additional metal binding sites to increase the quantity of metal atoms released or picked up through administration of metal containing and/or metal- free domains.
  • Alteration of the primary structures to form analogs may also alter the cofactor binding sites of the domains. Alteration of the cofactor binding sites enhances or diminishes the effects of the cofactors on the metal uptake and release of the analogs.
  • the domains of the present invention then can be tuned so as to optimize the metal binding to suit a particular therapeutic need.
  • the domains of the present invention also include derivatives, where such derivatives are domains that are bound to another atom, molecule or ion, where such other molecule or ion is either monomeric or polymeric, and where this other molecule or ion is not itself a domain.
  • the domains of the present invention may be metal-free or may contain one or more metal atoms.
  • a domain of the present invention is derivatized so as to be bound to an antibody or to a cellular antigen to target particular sites for release or uptake of metal ions at that site.
  • derivatization is well known to those skilled in the art.
  • a domain of the present invention includes one or more probe characteristics.
  • probe characteristics useful in the invention are incorporation of one or more radioactive metals into a domain, isotopic substitution of one or more atoms in the domain, fusion to a domain of one or more of a fiuorophore, an NMR contrast reagent, or a Foerster radiation-less energy transfer reagent, and arsenoazo modification of amino acid residues such as tyrosine (B. L. Vallee et al., Biochem. 11, 2584, 1972.).
  • probe characteristics and methods for their incorporation into proteins are well known to those skilled in the art.
  • Another embodiment of the present invention is a method for synthesis of the purified domains of the present invention in high yield and purity.
  • Prior syntheses of the ⁇ and ⁇ domains of metallothionein and other cysteine-rich polypeptides have employed N-alpha-t-boc-S-(4-methylbenzyl)-L-cysteine and N-alpha-fmoc-S-benzyl-L-cysteine. These syntheses have provided the desired polypeptides in very low yield (3 percent) and only 90 percent purity.
  • Fmoc-Cys(Acm) N-alpha-finoc-S-acetimidomethyl-L-cysteine
  • Fmoc-Cys(Acm) N-alpha-finoc-S-acetimidomethyl-L-cysteine
  • N-alpha-t-boc-S - (4-methylbenzyl)-L-cysteine or N-alpha-fmoc-S-benzyl-L-cysteine permits solid-phase synthesis of the desired polypeptides in about 90 percent yield, and because of the large quantity so obtained, in as high a purity as is desired for a given purpose.
  • Fmoc-Cys(Acm) is available commercially from Calbiochem-Novabiochem Corp. (San Diego, California).
  • alpha amino acids are selected from the group consisting of amino acids with aliphatic group containing side chains wherein said aliphatic group is hydrogen or alkyl, amino acids with aromatic group containing side chains, amino acids with sulfur group containing side chains wherein said sulfur group is a thiol or a thiol ether, amino acids with hydroxyl group containing side chains, amino acids with amine group containing side chains, amino acids with guanidinium group containing side chains, amino acids with carboxylate group containing side chains, and amino acids with amide group containing side chains, wherein the alpha amino groups are protected with a protecting group selected from the group consisting of Fmoc, t-Boc, and CBZ, the carboxylate groups are protected with a protecting group selected from the group consisting of t-butyl ester and benzyl ester, the hydroxyl groups are protected with a
  • step (b) cleaving the peptide synthesized in step (a) from the solid support and removing the non-acetimidomethyl protecting groups;
  • step (c) purifying the peptide obtained from step (b); (d) precipitating the peptide obtained from step (c);
  • the method step (a) is accomplished using an automated solid- phase synthesizer.
  • the domain is the alpha domain of metallothionein or the beta domain of metallothionein.
  • the alpha amino groups are protected with an Fmoc protecting group
  • the carboxylate groups are protected with a t- butyl ester protecting group
  • the hydroxyl groups are protected with a t-butyl ether protecting group
  • the amine groups are protected with a t-Boc protecting group.
  • the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; and the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid.
  • An example of such a gel is Sephadex 25.
  • the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.
  • the domain synthesized according to the method is the alpha domain of metallothionein or the beta domain of metallothionein; and wherein the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t-butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group; wherein the synthesizing step (a) is accomplished using a solid-phase synthesizer wherein the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; wherein the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydr
  • amino acids with aliphatic group containing side chains wherein said aliphatic group is hydrogen or alkyl include but are not limited to glycine (Gly, G), alamne (Ala, A), Valine (Val, V), Leucine (Leu, L), isoleucine (He, I), and proline (Pro, P).
  • amino acids with aromatic group containing side chains include but are not limited to phenylalanine (Phe, F), tryptophan (Tip, W), and histidine (His, H).
  • amino acids with sulfur group containing side chains wherein said sulfur group is a thiol or a thiol ether include but are not limited to cysteine (Cys, C) and methionine (Met, M).
  • amino acids with hydroxyl group containing side chains include but are not limited to tyrosine (Tyr, Y), serine (Ser, S) and threonine (Thr, T).
  • An example of an amino acid with an amine group containing side chain includes but is not limited to lysine (Lys, K).
  • An example of an amino acid with a guanidinium group containing side chain includes but is not limited to arginine (Arg, R).
  • amino acids with carboxylate group containing side chains include but are not limited to aspartic acid (Asp, D) and glutamic acid (Glu, E).
  • amino acids with amide group containing side chains include but are not limited to asparagine (Asn, N) and glutamine (Gin, Q).
  • Lys(boc)-Wang resin p-benzyloxybenzyl resin, Bachem, Switzerland
  • All amino acids used in the synthesis were N-alpha-frnoc protected, and the side-chain functional groups were protected as follows: O-tert-butylaspartic acid, O-tert-butylglutamic acids, O-tert- butylserine, O-tert-butylthreonine, lysine(Boc), and Cys(Acm); all protected amino acids were obtained from Bachem. Each protected amino acid was added to the synthesizer in
  • the purity of the peptides was determined by electrospray mass specfrometry and was higher than 93 %.
  • metal inhibitory sites typically are nanomolar, while those of catalytic metal sites are typically in the picomolar range.
  • metal-inhibited enzymes cannot be identified by the criteria which were established to recognize metalloenzymes with catalytic or structural metal atoms whose biological activity is a direct function of their total metal content, but may be identified by simple assays of their activity in the presence and absence
  • Metal-free domains prove to be very effective agents for the removal of zinc and other metals from such inhibitory sites in metal-inhibited enzymes, and metal-containing domains release metals to provide inhibition of these enzymes.
  • metal-free and metal-containing domains are useful for controlling enzymatic reactions in industrial,
  • Control of enzymatic reactions for purposes of the present invention means the ability to selectively increase or decrease the rate of the reaction converting a substrate to a product, catalyzed by the enzyme.
  • Enzyme reaction rates are expressed by those skilled in the art using a variety of terms, and these terms may depend on whether or not the reaction in question obeys particular rate laws. For purposes of this invention, the reaction rates are expressed in terms of a ratio of modified enzyme turnover rates in the presence and absence of an added domain of the present invention.
  • the normal (unmodified) enzyme turnover number is the number of substrate molecules converted into product by an enzyme molecule in a unit of time when the enzyme is fully saturated with substrate.
  • the modified enzyme turnover number used to express the reaction rates of the present invention is the number of substrate molecules converted into product by an enzyme molecule in one second under the particular reaction conditions employed in a process of interest.
  • An increase in the reaction rate due to addition of a metal-free or metal-containing domain of the present invention is expressed as a ratio of the modified enzyme turnover number in the presence of added domains divided by the modified enzyme turnover number in the absence of any added domains of the present invention.
  • a decrease in the reaction rate due to addition of a metal-free or metal-containing domain of the present invention is expressed as the ratio of the modified enzyme turnover number in the absence of added domains divided by the modified enzyme turnover number in the presence of any added domains of the present invention.
  • the change in reaction rate is called a factor, where the factor in the preceding examples is equal to 1.1.
  • a method for controlling an enzymatic reaction susceptible to such control, the reaction comprising conversion using the enzyme of a substrate to a product; the method comprising adding an amount of a composition comprising a domain to a reaction mixture comprising an enzyme; wherein the amount is sufficient to effect a change in a rate of the reaction, wherein the change is selected from the group consisting of an increase in the rate of reaction by a factor of at least about 1.1 and a decrease in rate of reaction by a factor of at least about 1.1; wherein the enzyme comprises an inhibitory site that binds a metal cation; wherein the domain is added to the reaction mixture at a time selected from the group consisting of a time before which the substrate is available to the enzyme the and a time during which the substrate is available to the enzyme; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of
  • the domain is the alpha domain of metallothionein or the beta domain of metallothionein.
  • the domain is an analog of the alpha domain of metallothionein wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
  • one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids
  • one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids.
  • a more prefe ⁇ ed embodiment uses an analog of the alpha domain of metallothionein where the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein by fusion of one primary sequence of the beta domain of metallothionein to the N-terminal end of the alpha domain of metallothionein.
  • the domains useful in control of enzymatic reactions are preferably purified.
  • Purified domains comprise no more than 5 percent impurities arising from the synthesis and isolation of the domain, and more preferably, no more than 2 percent impurities arising from the synthesis and isolation of the domain.
  • Other purified compositions useful in the enzyme control methods of the present invention use compositions where the domain comprises at least 95 % of the total protein in the composition, and preferably, where the domain comprises at least 98 % of the total protein in the composition.
  • the alpha and beta domains of MT have different binding affinities for zinc.
  • the beta domain releases zinc faster than the alpha domain.
  • the alpha domain is used in embodiments wherein zinc sequestration is desired, and the beta domain is used in embodiments wherein zinc delivery is desired.
  • Enzyme processes are important in various industries. These industries include but are not limited to food and beverage industries such as sweetening, baking, brewing, oil and shortening, cheesemaking, distilling, meat-packing, and fruits and vegetables (particularly juice), household and industrial cleaning products industries, particularly in laundering and stain removal applications, and in the fine and commodity chemical industries.
  • food and beverage industries such as sweetening, baking, brewing, oil and shortening, cheesemaking, distilling, meat-packing, and fruits and vegetables (particularly juice)
  • household and industrial cleaning products industries particularly in laundering and stain removal applications, and in the fine and commodity chemical industries.
  • glucose isomerases may be inhibited by the following metal cations: Ag(I), Ni(II), Hg(II), Fe(II), Zn(II), Ca(II), Al(III), Ga(II), Ba(II), As(III), and Cu(I, II).
  • metal cations Ag(I), Ni(II), Hg(II), Fe(II), Zn(II), Ca(II), Al(III), Ga(II), Ba(II), As(III), and Cu(I, II).
  • metal cations Ag(I), Ni(II), Hg(II), Fe(II), Zn(II), Ca(II), Al(III), Ga(II), Ba(II), As(III), and Cu(I, II).
  • amylolytic enzymes are used in sweetener industry, in brewing, in baking, and in distilleries.
  • Alpha amylases have calcium binding sites, where Ca(II) provides conformational stability for the active site.
  • a method for controlling reactions that use amylolytic enzymes comprising adding to the reaction one or more domains of the present invention.
  • the domain is metal-free.
  • proteases are used in meat tenderizing, texturing, beer chill-proofing, and cheese manufacturing.
  • Subtilsins have divalent Ca(II) binding sites which serve to stabilize conformations.
  • the action of papains and calpains is also know to be dependent on Ca(II).
  • a method for controlling reactions that use protease enzymes comprising adding to the reaction one or more domains of the present invention.
  • the domain is metal-free.
  • lipases are used in the oil and shortening industries to hydrolyze triglyceryl esters. Some lipases require a Ca(II) cofactor which binds in the active site.
  • a method for controlling reactions that use lipase enzymes comprising adding to the reaction one or more domains of the present invention.
  • the domain is metal- free.
  • Pectate lyase requires Ca(II) for activity; with pectinesterase, low concentrations of metal cations (e.g. Ca(II)) tend to enhance activity, particularly that involved in ripening.
  • a method for controlling reactions that use pectase enzymes comprising adding to the reaction one or more domains of the present invention.
  • the domain is metal-free.
  • xylose isomerases are used in production of high-fructose corn syrup. Some xylose isomerases require Mg(II), Mn(II), Ni(II) or Co(II) for activity.
  • a method for controlling reactions that use xylose isomerase enzymes comprising adding to the reaction one or more domains of the present invention.
  • the domain contains a metal selected from the group consisting of Mg(II), Mn(II), Ni(II) and Co(II).
  • the domain is metal-free.
  • Enzymatic reactions are important for the efficacy of certain products sold for household and industrial uses, particularly for cleaning applications.
  • Household and industrial cleaners and detergents employ proteases, and sometimes lipases to remove protein and lipid deposits.
  • a method for controlling an enzymatic reaction used in a cleaning application comprising adding one or more domains of the present invention to a composition comprising an enzymatic cleaning agent.
  • Enzymatic reactions are used as part of clinical and industrial assays.
  • many assays used to detect disease states (a non-limiting example being diabetes) and levels of biologically relevant molecules in body fluids (a non-limiting example being pregnancy tests) and industrial mixtures (non-limiting examples being foods and beverages) use enzymatic reactions.
  • a method for controlling an enzymatic reaction used in a clinical or industrial assay comprising adding one or more domains of the present invention to a composition used in the assay, the assay comprising an enzymatic reaction.
  • Enzymatic reactions are used as part of chemical synthesis and biotechnological transformations.
  • immobilized enzymes and whole cells are used in small and large scale synthesis of organic molecules and for inducing expression of biologicals in biotechnological applications.
  • a method for controlling an enzymatic reaction used in a organic chemical synthesis comprising adding one or more domains of the present invention to a composition used in the synthesis.
  • the product of the reaction is an organic molecule selected from the group consisting of an intermediate in an organic synthesis and a final product of an organic synthesis.
  • the method of the present invention is used in a biotechnological reaction where the product is a biomolecule selected from the group consisting of an intermediate in a biochemical process and a final product of a biochemical process.
  • T thionein
  • Rabbit liver thionein was prepared from cadmium MT-1 (Vasak, M.,
  • Buffers and stock solutions were purged with nitrogen gas for 30 min. Enzymes were brought into 20 mM Hepes-Na + , pH 7.5 ("buffer") by passage through a PD-10 gel filtration column (Pharmacia). Ultrapure MgSO 4 , MgCl 2 , ZnCl 2 , ZnSO 4 and KC1 were from Johnson Matthey Chemicals Ltd., England. Ac-DEVD-AMC was from Pharmingen.
  • Enolase (rabbit muscle) from Sigma which contained no detectable zinc was diluted to a final concentration of 10 nM in buffer containing 2 mM MgCl 2 and activity measured spectrophotometrically at 237 nm for 2 min after the reaction was started with D- 2-phosphoglyceric acid at a final concentration of 1 mM. Analogous experiments were performed with enolase (yeast) from Sigma.
  • Fructose 1,6-Diphosphatase Fructose 1 ,6-diphosphatase (rabbit liver) from Sigma which contained no detectable zinc was diluted to a final concentration of 5 nM in buffer containing 1 mM MgCl 2 . Enzyme assays were initiated by addition of D-fructose 1,6- diphosphate to a final concentration of 100 ⁇ M, quenched after 15 min and the phosphate released titrated with the molybdenum blue method (Tashima, Y. and Yoshimura, N., 1975, J. Biochem. (Tokyo) 78, 1161-1169). This time interval is in the linear range of phosphate production.
  • Glyceraldehyde 3-Phosphate Dehydrogenase (rabbit muscle) from Sigma was diluted to final concentrations of 2.5 and 10 nM in buffer containing 30 mM Na ⁇ AsO ⁇ Glyceraldehyde 3-phosphate (30 mM stock solution) and NAD + (15 mM stock solution) were added to final concentrations of 500 and 250 ⁇ M, respectively and the reaction monitored spectrophotometrically at 340 nm for 2 min.
  • Aldehyde Dehydrogenase Yeast aldehyde dehydrogenase from Sigma was diluted to a final concentration of 155 nM in buffer containing 100 mM KC1. Acetaldehyde (40 mM stock solution) and NAD + (5 mM stock solution) were added to final concentrations of 2 and 0.5 mM, respectively and the reaction monitored spectrophotometrically at 340 nm for 2 min.
  • Protein Tyrosine Phosphatase The recombinant protein tyrosine phosphatase from Calbiochem used for our studies is a truncated form of the human T-cell protein tyrosine phosphatase with an 1 lkDa deletion at the C-terminus (Cool, D. E., Tonks, N. K., Charbonneau, H, Walsh, K. A., Fischer, E. H. and Krebs, E. G., 1989, Proc. Natl. Acad. Sci. USA 86, 5257-5261). Phosphatase was diluted to a final concentration of 20 nM in buffer. An aliquot of 4-nitrophenyl phosphate was added (100 mM stock solution) to give a final concentration of 1 mM and the formation of 4-nitrophenolate was monitored
  • Caspase-3 Recombinant human caspase-3 from Pharmingen was diluted to a final concentration of 1.7 nM in buffer.
  • Ac-DEVD-AMC (1.12 mM stock solution) was added to a final concentration of 22.4 ⁇ M and the formation of AMC (amino-4-methylcoumarin) monitored fluorimetrically for 5 min (excitation wavelength: 380 nm, emission wavelength:
  • FluoroMax-2 Instruments S.A., Inc. fluorimeter (Nicholson, D. W., Ali, A., Thornberry, N. A., Villancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T.- T., Yu, V. L. and Miller, D. K., 1995, Nature 376, 37-43).
  • Alcohol dehydrogenase Alcohol dehydrogenase (horse liver) from Boehringer
  • Alcohol dehydrogenase was diluted to a final concentration of 100 nM in buffer. Enzymatic activity was measured by following the formation of NADH spectrophotometrically at 340 nm for 2 min after adding ethanol (6 M stock solution) and NAD + (25 mM stock solution) to final concentrations of 330 and 1 mM, respectively.
  • Carbonic Anhydrase (bovine erythrocytes) from Sigma contained 0.93 zinc atom per monomer. Carbonic anhydrase was diluted to a final concentration of 250 nM in buffer. Enzymatic activity was measured spectrophotometrically at 400 nm for 2 min after adding 4-nitrophenyl acetate (20 mM stock solution in acetonitrile) to a final concentration of 0.5 mM (Henkens, R. W. and
  • Glutathione Peroxidase Glutathione peroxidase (bovine erythrocytes) from Sigma was diluted to a final concentration of 2.4 nM in buffer. Enzymatic activity was measured spectrophotometrically at 260 nm for 1 min after adding t-butyl hydroperoxide (50 mM stock solution) and glutathione (0.2 M stock solution) to final concentrations of 5 and 2
  • Glutathione Reductase Glutathione reductase (yeast) from Sigma was diluted to a final concentration of 8 nM in buffer. Enzymatic activity was measured spectrophotometrically at 340 nm after adding NADPH (4.5 mM stock solution) and glutathione (5 mM stock solution) to final concentrations of 50 and 2 ⁇ M, respectively.
  • the IC 50 values for zinc inhibition of the enzymes studied save rabbit enolase are below 250 nM. Since inhibition is in the nanomolar range, the IC 50 values are a function of the enzyme concentration which on occasion had to be lowered significantly, sometimes to the limit of sensitivity of the assay. Thus these only reflect upper limits and the true inhibition constants are likely even lower.
  • the inhibition of caspase-3 turns out to be most notable among those examined since it reflects a virtual 1:1 stoichiometric reaction: At equimolar concentrations of enzyme (1.7 nM) and zinc only 50% of the initial enzymatic activity remains (Figure 1). Again, the 5-fold higher enzyme concentration employed in a previous study clearly required a higher zinc concentration for inhibition (Perry, D.
  • Enzyme (nM) IC 50 measured, nM IC 50 reported, nM caspase-3 (1.7) ⁇ 10 100 1 ⁇ fructose 1,6-diphosphatase (5) 100 300 2) glyceraldehyde 3-phosphate 150 — dehydrogenase (2.5) aldehyde dehydrogenase (155) 154 — tyrosine phosphatase (20) 200 100,000 3) enolase (yeast) (10) 4) 250 2,500 5) 1) Perry, D. K., Smyth, M. J., Stennicke, H. R., Salvesen, G. S., Duriez, P., Poirier, G. G. and Hannun, Y. A., 1997) J. Biol. Chem. 272, 18530-18533
  • a catalytic cysteinyl residue is known to be characteristic of glyceraldehyde 3- phosphate dehydrogenase, aldehyde dehydrogenase, protein tyrosine phosphatase, and caspase-3, we have searched for other enzymes that might have potential cysteine (or selenocysteine) ligands at their active site. Unlike the above enzymes, nanomolar concentrations of zinc do not inhibit glutathione peroxidase from bovine erythrocytes (catalytic selenocysteine) or glutathione reductase from yeast (catalytic vicinal cysteines).
  • glutathione peroxidase In the presence of 1 ⁇ M zinc, glutathione peroxidase (2.4 nM) loses only 15% of its activity.
  • the IC 50 value for glutathione reductase (8 nM) is about 10 ⁇ M.
  • the presence of a cysteine residue at the catalytic site is not sufficient to provide an inhibitory site: the active site of enzymes with potential metal binding ligands may be designed in such a way that they are protected from inhibition by zinc or other metal ions.
  • T binds zinc very tightly to form zinc/sulfur clusters that do not exist in the inanimate world. Thus far T has not proven to be
  • Zinc is known to inhibit multiple biological processes demonstrating that its cellular concentration must be controlled carefully and kept well below the micromolar range to preserve the activity of zinc metalloenzymes and others critical to metabolism. This is accomplished in part by the cellular MT/T system. If free zinc concentrations were to rise into the micromolar range, numerous enzymatic processes could be compromised.
  • the binding constants for zinc in T-reversible inhibitory sites of enzymes on the one hand and in catalytically active zinc metalloenzyme sites on the other seem to establish the conditions for the availability of cellular zinc, while demonstrating the need for its efficient regulation. Dissociation of zinc from zinc metalloenzymes occurs below 10 "10 M while zinc inhibition becomes sigmficant above 10 "8 M. The definition of this upper set-point in the nanomolar range depends on the exact magnitudes of inhibition constants.
  • T expression is regulated by a metal response element binding factor (MTF-1) which in turn is itself activated by zinc (Radtke, F., Heuchel, R, Georgiev, O., Hergersberg, M., Gariglio, M., Dembric, Z. and Schaffner, W., 1993, EMBO J. 12, 1355- 1362).
  • the chromophoric properties of the metal-thiolate complexes of MT provide a means to assess the stability of the metal-protein complex by pH titrations.
  • the point of half-maximum absorbance of the alpha cluster is lower than that for the beta cluster, demonstrating that zinc and cadmium bind more tightly to the alpha cluster than the beta cluster ( Figure 4).
  • both human Zn 7 -MT-2 and cadmium MT-2 are stabilized as compared to the sum of the individual domains ( Figure 5).
  • proton titrations show the alpha cluster to be more thermodynamically stable than the beta cluster.
  • the beta cluster Unlike the PAR system, the release of zinc from the beta cluster and its transfer to apo-SDH is almost ten times faster than that from the alpha cluster, suggesting different mechanisms of zinc transfer to these two acceptors. In both cases, the beta cluster releases zinc faster than the alpha cluster. Chemical reactivity. Under pseudo-first-order rate conditions when the concentration of the disulfide 5,5'-Dithiobis 2-nitrobenzoic acid (DTNB) is less than one equivalent per sulfur ([DTNB] «[cluster thiols]), the beta cluster reacts faster than the alpha cluster (Figure 8).
  • DTNB disulfide 5,5'-Dithiobis 2-nitrobenzoic acid
  • the whole MT molecule is less reactive than its individual clusters, demonstrating that domain interactions in the whole molecule affect the reactivity of MT in addition to its effect on the structure of MT.

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Abstract

The present invention relates to the alpha and beta domains of metallothionein and analogs thereof, their synthesis, and methods for controlling in vitro and industrial enzymatic reactions using them. Purified metal-free and metal-containing alpha and beta domains of metallothionein and analogs thereof are provided as described below. A high yield method of synthesis and purification is also provided for the metal-free and metal containing alpha and beta domains of metallothionein and analogs thereof. Finally, methods for controlling in vitro and industrial enzymatic reactions are provided wherein the enzymatic processes are controlled by transfer or removal of a metal cation, to or from an enzyme metal-inhibitory site using the alpha and beta domains of metallothionein and analogs thereof.

Description

A METHOD FOR CONTROLLING AN ENZYMAΗC REACTION BY THE ALPHA AND BETA DOMAINS OF METALLOTHIONEIN
1. INTRODUCTION
5 The present invention relates to the alpha and beta domains of metallothionein and analogs thereof, their synthesis, and methods for controlling in vitro and industrial enzymatic reactions using them. Purified metal-free and metal-containing alpha and beta domains of metallothionein and analogs thereof are provided as described below. A high yield method of synthesis and purification is also provided for the metal-free
1 o and metal-containing alpha and beta domains of metallothionein and analogs thereof. Finally, methods for controlling in vitro and industrial enzymatic reactions are provided wherein the enzymatic processes are controlled by transfer or removal of a metal cation, to or from an enzyme metal-inhibitory site using the alpha and beta domains of metallothionein and analogs thereof.
15
2. BACKGROUND OF THE INVENTION a. METALLOTHIONEIN
Metallothionein (MT) was discovered in 1957 (Margoshes, M. and Nallee, B. L., 1957, J. Am. Chem. Soc. 79, 4813). By all counts it is a most unusual and
20 unconventional protein (Nallee, B.L., 1979, Experientia Suppl. 34, 19-40; Vallee, B.L., 1987, Experientia Suppl. 52, 5-16; Nallee, B. L., 1991, Meth. Enzymol. 205, 3-7; Vallee, B.L. and Maret, W., 1993, in Metallothionein III, eds. Suzuki, K. T., Imura, Ν. and Kimura, M. (Birkhauser, Basel), 1; Nallee, B.L., 1995, Νeurochem. Interntl. 27, 23). One third of its 60+ amino acids are cysteines and eight are lysines. It contains neither aromatic amino
25 acids nor histidine. MT usually binds seven zinc atoms, but it can also contain copper, cadmium, iron and traces of other metals. In an evolutionary sense it is a very old protein.
The composition of the two major MT isoproteins has remained conserved over time. MT-1 and MT-2 are the two prevalent forms, which are expressed, but whose physiological functions are unknown. MT-3 was discovered only recently in human brains
30 (Uchida, Y., Takio, K., Titani, K., Ihara, Y., and Tomonaga, M., 1991, Neuron 7, 337). Its discovery was based on the fact that it inhibits the growth of neurons. Thus far, it is the only MT that is known to exhibit such a specific biological function. This isoform contains zinc and copper(I), but not cadmium or other metals.
The number of genes that code for human MTs could be as high as 17. Multiple
35 factors (among them members of the nuclear hormone receptor family, interferons, inducers of the acute phase response, and metalloregulatory proteins) affect tissue- and isoprotein- specific gene expression. In addition there are numerous other agents that induce it but whose signaling pathways remain obscure. Thionein, the apoform of MT has never been isolated as such from any biological material. Apparently, upon its formation, it instantaneously combines with zinc, whose "free" concentration in the cell has been reported to be exceedingly low, i.e. in the nanomolar to picomolar range.
b. THIONEIN
While Metallothionein has been studied very extensively since its discovery the biochemistry of thionein (T), its apoprotein, has received relatively little experimental attention thus far. Efforts to demonstrate its endogenous production have consistently failed, in some measure owing to its lack of any appropriate spectroscopic property that could be guides to its isolation. A series of manuscripts from this laboratory (Maret, W. and Nallee, B. L., 1998, Proc. Νatl. Acad. Sci. USA 95, 3478-3482; Jiang, L-J., Maret, W. and Nallee, B. L., 1998, Proc. Νatl. Acad. Sci. USA 95, 3483-3488; Jacob, C, Maret, W. and Nallee, B. L., 1998, Proc. Νatl. Acad. Sci. USA 95, 3489-3494) have indicated its transient existence and generation at high local concentrations at the instant of its formation, emphasizing its importance as an endogenous and potent zinc chelating agent, effective at exceedingly low cellular concentrations. We are unaware of any analogous biological substance with corresponding properties. T avidly binds metal ions and is highly susceptible to proteolytic digestion and oxidation. T is a very efficient biological chelating agent for zinc. It suppresses the DΝA-binding capacity of zinc finger transcription factors in vitro by sequestering zinc and removing it from their structural sites (Zeng, J., Heuchel, R., Schaffher, W. and Kagi, J. H. R., 1991, FEBS Lett. 279, 310-312; Zeng, J., Nallee, B. L. and Kagi, J. H. R, 1991, Proc. Νatl. Acad. Sci. USA 88, 9984-9988; Roesijadi, G., Bogumil, R., Vasak, M. and Kagi, J. H. R, 1998, J. Biol. Chem. 273, 17425-17432.). We have previously established conditions to identify, isolate and store T so that it completely retains its function including cluster formation through binding by its sulfhydryl groups (Jacob, C, Maret, W. and Vallee, B. L., 1998, Proc. Νatl. Acad. Sci. USA 95, 3489-3494). We have also shown that zinc is transferred from MT to the apoforms of zinc metalloenzymes and that T is indeed transiently formed in situ during this process (Jiang, L- J., Maret, W. and Vallee, B. L., 1998, Proc. Νatl. Acad. Sci. USA 95, 3483-3488; Jacob, C, Maret, W. and Vallee, B. L., 1998, Proc. Νatl. Acad. Sci. USA 95, 3489-3494) For the reverse reaction, T itself does not remove significant amounts of zinc from zinc metalloenzymes. Instead, agents such as glutathione or citrate, which can bind zinc themselves, but do not remove it from the active site of the zinc metalloenzyme, can serve in the transfer process (Jacob, C, Maret, W. and Vallee, B. L., 1998, Proc. Νatl. Acad. Sci. USA 95, 3489-3494).
c. ZINC AS AN INHIBITOR OF ENZYMES
In addition to its catalytic role in more than 300 zinc metalloenzymes and its structural role in an even greater number of non-enzymatic proteins, zinc also is a known inhibitor of enzymes in general, including zinc metalloenzymes. In 1960 zinc binding experiments demonstrated a second, non-active zinc site in carboxypeptidase A (Coleman, J. E. and Vallee, B. L., 1960, J. Biol. Chem. 235, 390-399). Twenty years later this was established as an inhibitory site which was later on shown to be located in the vicinity (3.3 A) of the catalytic zinc atom and to inhibit the enzyme with an apparent K] value of 24 μM (Larsen, K. L. and Auld, D. S., 1991, Biochemistry 30, 2613-2618). If it is taken into account that ZnOH+ is the inhibitory species, the actual Kτ value is 0.7 μM. Larsen and Auld (Larsen, K. L. and Auld, D. S., 1991, Biochemistry 30, 2613-2618; Larsen, K. L. and Auld, D. S., 1989, Biochemistry 28, 9620-9625) proposed that the inhibitory zinc atom is bound to Glu-270 and binds both an anion and a water molecule, and that a hydroxide bridges the inhibitory and catalytic zinc atoms as has been confirmed by X-ray diffraction (Gomez-Ortiz, M., Gomis-Ruth, F. X., Huber, R. and Aviles, F. X, 1997, FEBS Lett. 400, 336-340; Bukrinski, J. T., Bjerrum, M. J. and Kadziola, A., 1998, Biochemistry 37, 16555- 16564). This particular inhibitory site has proven to be but one of many such inhibitory zinc sites found in non-metalloenzymes. These have also been recognized recently in neurotransmitter receptors of the central nervous system (Chen, N., Moshaver, A. and Raymond, L. A., 1997, Mol. Pharmacol. 51, 1015-1023).
The K, values for zinc inhibition of bacterial adenylosuccinate synthase and 6-phosphogluconate dehydrogenase are 29 and 21 nM, respectively (Kang, C. and Fromm, H. J., 1995, J. Biol. Chem. 270, 15539-15544; Niehaus, W. G., Richardson, S. B. and Wolz, R. L., 1996, Arch. Biochem. Biophys. 333, 333-337). Their eukaryotic counterparts, however, have not been examined as yet in this regard.
i. Protein Phosphorylation and Dephosphorylation Protein phosphorylation and dephosphorylation plays a central role in metabolism and cellular signaling. Protein kinases (PK) and protein phosphorylases (PP) play complimentary roles in signal transduction from the extracellular environment to the cytosol. For example, insulin receptor transduction involves tyrosine kinase domain activation on binding of insulin, with subsequent phosphorylation of target cytosol proteins by the activated receptor.
Protein phosphatases remove inorganic phosphate from phosphorylated proteins, and thus attenuate signals involving phosphorylated proteins. Protein-tyrosine phosphatases (PTP) and protein serine/threonine phosphatases (PSP) are thus central to regulation of cellular processes. Furthermore, at least one oncogene is known to code for a protein-tyrosine kinase (PTK). Intrinsic PTP activity is known to be two to three orders of magnitude greater than PTK activity, and thus the cellular levels of endogenous phosphorylated tyrosine residues is ordinarily low. All PTPs share a common signature motif from bacterial to mammalian enzymes.
d. THE α AND β DOMAINS OF METALLOTHIONEIN MT was discovered 40 years ago. The 3D structure of MT has been solved both by X-ray crystallography (Robbins, A. H. and Stout, C. D., 1991, Meth. Enzymol. 205, 485) and NMR spectroscopy (Wuthrich, K., 1991, Meth. Enzymol. 205, 502). The protein has the shape of a dumb-bell and envelops the metals which it contains in two separate domains (termed "α" and "β") in a manner which effectively shields them from the environment. The amino acid sequence of the two domains has been reported, as well (Winge et al., J. Biol. Chem., 1984, 259, 11419). It is most remarkable that the metals are arranged in cluster structures unique to biology. In one cluster four metal atoms are bound to eleven cysteines, five of which bridge the metals, and the other has three metal atoms and nine cysteines with three bridges. The metal-free domains have previously been prepared by several methods, none of which provide these domains in high yield in pure form. Partial enzymatic digestion of the native protein followed by metal removal has been reported (Winge, D. R.; Miklossy, K.-A., J. Biol. Chem. 1982, 257, 3471; Nielson, K. B.; Winge, D. R., J. Biol. Chem., 1984, 259, 4941). These domains have also been expressed in E. coli. Solid-phase synthesis of the α and β domains of MT-2 has been reported, which synthesis provided the domains in 90 to 95 percent purity in only 3 to 4 percent yield (Kull, F. A., et al., J. Am. Chem Soc. 1990, 112, 2291).
The metal-binding properties of the individual domains has been studied with respect to Cu(I), Zn(II), Cd(II), Ag(I), and Co(II). See, for example, Nielson, K. B., Winge, D. R., J. Biol. Chem., 1985, 260, 8698; Good, M., Vasak, M., Biochemistry, 1986, 25, 3328; George, G. N., et al., J. Inorg. Biochem., 1986, 27, 213; Zelazowski, A. J., et al., J. Biol. Chem., 1984, 259, 12950; Stillman, M. J., et al., J. Biol. Chem., 1987, 226, 4538; Kull, F. A., et al, J. Am. Chem Soc. 1990, 112, 2291.
e. THE BIOLOGICAL IMPORTANCE OF TRACE ELEMENTS
Iron, copper, and zinc are known to be essential to most or all forms of life. Additionally, vanadium, chromium, manganese, cobalt, nickel, arsenic, selenium, molybdenum and tungsten are known to be essential to some or many life forms. Many of these metals function as part of metalloproteins, either structurally, or in the case of some metalloenzymes, catalytically. Furthermore, metals such as lithium are known to have pharmacological effects.
Catalytic functions of zinc in enzymes and its structural functions in zinc finger proteins have been documented amply (Vallee, B. L. and Falchuk, K. F., 1993, Physiol. Rev. 73, 79-118; Vallee, B. L. and Auld, D. S., 1993, Ace. Chem. Res. 26, 543). Zinc in MT is bound extremely tightly (KD about 10"13 M) (Kagi, J. H. R., 1993, in Metallothionein III, eds. Suzuki, K. T., Imura, N. and Kimura, M. (Birkhauser, Basel), p. 29).
3. DEFINITIONS
As used herein, the following terms shall have the meanings indicated: MT = metallothionein;
GSH = glutathione; T = thionein;
EDTA = ethylenediaminetetraacetic acid; finoc = 9-fluorenylmethyloxycarbonyl; t-boc or boc = tertiary-butyloxycarbonyl;
CBZ = carbobenzoxy.
As used herein, the term compound means any molecule, salt, metal, or any other combination of one or more atoms, including but not limited to covalently bonded molecules, ionic materials, metallic materials, crystalline materials, atoms or molecules or ions in solution, atoms or molecules or ions in the gas phases, and combinations of any of the preceding.
As used herein, (unless otherwise specified) the term metallothionein includes all of the isoforms of metallothionein- 1, metallothionein-2, metallothionein-3, and metallothionein-4; furthermore the primary sequence of such metallothionein can correspond to that of any species known to produce metallothionein.
As used herein, the term domain means a synthetic polypeptide synthesized by the procedures of the present invention, that (1) has the same primary structure as the alpha domain of metallothionein, (2) has the same primary structure as the beta domain of metallothionein, or (3) is an analogous polypeptide (hereinafter "analog") having a closely related primary structure to either the alpha or beta domain of metallothionein which analogous polypeptide is capable of binding metals in a cluster arrangement similar to the arrangements of the alpha or beta domain of metallothionein. The differences in primary sequences of the analogs as compared to the alpha or beta domains of metallothionein are
(a) substitution of one or more non-cysteine residues with different amino acids (either naturally occurring or non-naturally occurring amino acids, including but not limited to side-chain modified amino acids),
(b) repetition of, repetitions of, or combination of, the primary sequences of alpha and/or beta domains of metallothionein (with or without spacer sequences to separate the repeated or combined sequences) to provide larger polypeptides with more metal binding sites, 0 (c) one or more additional peptide residues grafted to the N-terminal and/or C- terminal end or ends of the alpha or beta domains of metallothionein, or (d) a combination of two or more of the substitution of residues, repetition/combination of sequences, and additional N-terminal and C- terminal residues described in the preceding subparagraphs (a), (b), and (c). 15 As used herein, the term domain also includes derivatives of domains. Unless otherwise specified, the term domain shall include metal-free and metal-containing domains, and for purposes of the present invention, selenium shall be considered to be included in the term metal.
As used herein, the term derivative means a molecule (including but not 0 limited to amino acids and peptides) chemically modified, for example by binding to that molecule another atom, molecule or ion, where such other molecule or ion is either monomeric or polymeric.
As used herein, the term sequence means two or more amino acids, and where there is more than one amino acid, the amino acids are covalently bonded through 5 one or more peptide bonds, with the number of peptide bonds being one less than the number of amino acids.
4. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Inhibition of caspase-3 by zinc. Caspase-3, 1.7 nM, was incubated with 30 various amounts of zinc. Activity was assayed after 5 min.
Figure 2. Protection and reactivation of glyceraldehyde 3-phosphate dehydrogenase with T in the presence of zinc. Glyceraldehyde 3-phosphate dehydrogenase, 10 nM, was incubated in buffer containing 30 mM Na2HAsO4 with various amounts of T introduced 5 min after ZnSO4 was added to a final concentration of 1 μM. Activities were assayed after 35 30 min.
Figure 3. Reactivation of zinc-inhibited glyceraldehyde 3-phosphate dehydrogenase with T. Glyceraldehyde 3-phosphate dehydrogenase, 10 nM, was incubated with ZnSO4, 1 μM, for 10 min in buffer containing 30 mM NajHAsO^ Enzyme activity was measured for 0.5 min, T, 250 nM, was then injected into the cuvet, and the measurement continued.
Figure 4. Titrations of zinc (A) and cadmium (B) reconstituted alpha (filled squares) and beta (open squares) domains vs. pH. Measurements were made on solutions containing 7 μM clusters and 0.1 M sodium perchlorate.
Figure 5. Comparison between the titrations of the whole human zinc (A) and cadmium (B) MT-2 molecules (filled squares) and the added titrations of the clusters (dashed lines). The titrations were performed on MT samples (7 μM) in 0.1 M sodium perchlorate.
Figure 6. Zinc transfer from alpha (filled squares) and beta (open squares) clusters to 4-(2-Pyridylazo)resorcinol (PAR). Clusters (2 μM) were incubated with PAR (200 μM) and the reaction swere followed spectrophotometrically.
Figure 7. Reconstitution of zinc-depleted sorbitol dehydrogenase (apo-SDH) with the clusters. The beta (A) and alpha (B) domain were incubated with apo-SDH (1.7 μM) at different ratios between zinc and apo-SDH.
Figure 8. Thiol reactivity of the clusters. The alpha (open squares) and beta (filled squares) domains (10 μM) were incubated with 4 μM 5,5'-Dithiobis 2-nitrobenzoic acid (DTNB) and the reactions followed spectrophotometrically. Inset: first-order replot of the data.
5. SUMMARY OF THE INVENTION
In a first embodiment, the present invention provides a method of synthesis and purification of a domain having a primary amino acid sequence comprising the following steps:
(a) synthesizing the domain using a solid support and at least two alpha amino acids having alpha amino groups, wherein the alpha amino acids are selected from the group consisting of amino acids with aliphatic group containing side chains wherein said aliphatic group is hydrogen or alkyl, amino acids with aromatic group containing side chains, amino acids with sulfur group containing side chains wherein said sulfur group is a thiol or a thioether, amino acids with hydroxyl group containing side chains, amino acids with amine group containing side chains, amino acids with guanidinium group containing side chains, amino acids with carboxylate group containing side chains, and amino acids with amide group containing side chains, wherein the alpha amino groups are protected with a protecting group selected from the group consisting of Fmoc, t-Boc, and CBZ, the carboxylate groups are protected with a protecting group selected from the group consisting of t-butyl ester and benzyl ester, the hydroxyl groups are protected with a protecting group selected from the group consisting of t-butyl ethers, and dimethylphosphate esters, the amine groups are protected with a protecting group selected from the group consisting of t-Boc and CBZ, and the thiol groups are protected with an acetimidomethyl group; (b) cleaving the peptide synthesized in step (a) from the solid support and removing the non-acetimidomethyl protecting groups;
(c) purifying the peptide obtained from step (b);
(d) precipitating the peptide obtained from step (c); and
(e) removing the acetimidomethyl protecting group with a solution comprising a silver(I) salt; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of a metallothionein, an analog of the alpha domain of metallothionein, and an analog of the beta domain of metallothionein; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids,
(ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and
(viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine,
(iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine,
(iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein,
(vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii). Preferably, the method step (a) is accomplished using an automated solid- phase synthesizer.
Also, preferably, the domain is the alpha domain of metallothionein or the beta domain of metallothionein.
In a prefeπed embodiment of the method, the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t- butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group.
In another preferred embodiment of the method, the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; and the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid.
In yet another preferred embodiment of the method, the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.
Most preferably, the domain synthesized according to the method is the alpha domain of metallothionein or the beta domain of metallothionein; and wherein the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t-butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group; wherein the synthesizing step (a) is accomplished using a solid-phase synthesizer wherein the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; wherein the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid; and wherein the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.
Preferably, all the alpha amino groups are protected with an Fmoc protecting group, all the side chain carboxylic acid groups are protected with a t-butyl ester protecting group, all side chain hydroxyl groups are protected with a t-butyl ether protecting group, and all side chain amine groups are protected with a t-Boc protecting group. Also, preferably, the cleavage of the peptide from the solid support and removal of the non- acetimidomethyl protecting groups is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid. Also, preferably, the purification by gel filtration chromatography is accomplished using a gel prepared from Sephadex-25 which comprises beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and preferably, the Sephadex gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid. Also, preferably, the removal of the acetimidomethyl protecting group is accomplished with a solution comprising silver(I) nitrate in acetic acid.
In another embodiment, the invention also provides the domains according to method of synthesis and purification. These domains may be metal containing or metal- free. Where metal containing, the metal may be selected from the group consisting of main group metals, transition metals, lanthanides, and actinides. Preferred metals of the invention are zinc, copper, gold, cadmium, iron, cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technetium, lanthanum, and combinations thereof. Most preferably, the metal is zinc.
The invention also provides compositions comprising a domain, wherein said domain comprises no more than 5 percent impurities arising from the synthesis and isolation of the domain, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine,
(iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine,
(iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv),
(v), (vi) and (vii); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of (i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein,
(vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii). Preferably, the domain of this composition comprises no more than 2 percent impurities arising from the synthesis and isolation of the domain.
The invention further provides a composition comprising a domain, wherein said domain comprises at least 95 % of the total protein in said composition, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine,
(v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids,
(ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine,
(vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii).
Preferably the domain of this composition comprises at least 98 % of the total protein in said composition.
The invention also provides compositions comprising a domain, wherein said domain comprises no more than 5 percent impurities arising from the synthesis and isolation of the domain, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of (i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iϋ) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine,
(v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and
(viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii). Preferably, the domain of this composition comprises no more than 2 percent impurities arising from the synthesis and isolation of the domain. The invention further provides a composition comprising a domain, wherein said domain comprises at least 95 % of the total protein in said composition, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids,
(ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and
(viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ϋ) substitution of one or more cysteine residues with selenocysteine,
(iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine,
(iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein,
(vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii). Preferably the domain of this composition comprises at least 98 % of the total protein in said composition.
In one embodiment of the present invention, a domain of the present invention includes one or more probe characteristics. Non-limiting examples of probe characteristics useful in the invention are incorporation of one or more radioactive metals into a domain, isotopic substitution of one or more atoms in the domain, fusion to a domain of one or more of a fluorophore, an NMR contrast reagent, or a Foerster radiation-less energy transfer reagent, and arsenoazo modification of amino acid residues such as tyrosine.
The domains may be formulated as pharmaceutical compositions. Such pharmaceutical compositions comprise one or more domains and further comprise one or more ingredients selected from the group consisting of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a wetting agent, a buffering agent, an emulsifying agent, and a binding agent. These compositions may also use domains wherein the domains are immobilized on a biologically-inert non-polymeric or polymeric support. This support may comprise, but is not limited to, a material selected from the group consisting of a polymeric support in a form selected from a bead, a fiber, and a sheet; paper; and cotton thread. Preferably, the polymeric support comprises a functionalized polystyrene.
In another embodiment, the invention provides methods for controlling enzymatic reactions susceptible to such control, the reactions comprising conversion of a substrate using the enzyme to a product; the method comprising adding an amount of a composition comprising a domain to a reaction mixture comprising an enzyme; wherein the amount is sufficient to effect a change in a rate of the reaction, wherein the change is selected from the group consisting of an increase in the rate of reaction by a factor of at least about 1.1 and a decrease in rate of reaction by a factor of at least about 1.1; wherein the enzyme comprises an inhibitory site that binds a metal cation; wherein the domain is added to the reaction mixture at a time selected from the group consisting of a time before which the substrate is available to the enzyme and a time during which the substrate is available to the enzyme; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids,
(ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ϋ) substitution of one or more cysteine residues with selenocysteine,
(iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine,
(iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii).
Preferably, the domain is the alpha domain of metallothionein or the beta domain of metallothionein. Also preferably, the domain is an analog of the alpha domain of metallothionein wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of (ii) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (ϋ) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids. Most preferably, the domain is an analog of the alpha domain of metallothionein whose primary amino acid sequence differs from the primary amino acid sequence of the alpha domain of metallothionein by fusion of one primary sequence of the beta domain of metallothionein to the N-terminal end of the alpha domain of metallothionein.
The domains useful in controlling enzymatic reactions may be purified. They may comprise no more than 5 percent impurities arising from the synthesis and isolation of the domain, and more preferably, no more than 2 percent impurities arising from the synthesis and isolation of the domain. For compositions containing purified domains for use in controlling enzymatic reactions, the domains comprise at least 95 % of the total protein in the composition, and preferably, at least 98 % of the total protein in the composition. For control of enzymatic reactions, the domains may be metal free or metal containing, and the domains may contain one or more metals selected from the group consisting of main group metals, transition metals, lanthanides, and actinides. Preferably, the metal containing domains will contain one or more metals selected from the group consisting of zinc, copper, gold, cadmium, iron, cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technetium, lanthanum, and combinations thereof, and most preferably, will contain zinc.
The enzymes of the reactions that may be controlled are preferably selected from the group consisting of glucose isomerases, amylolytic enzymes, proteases, lipases, pectases, and xylose isomerases. For control of glucose isomerases, the domain preferably contains at least one metal selected from the group consisting of Ag(I), Ni(II), Hg(II), Fe(II), Zn(II), Ca(II),Al(III), Ga(II), Ba(II), As(III), and Cu(I, II) and combinations of these metals. For control of amylolytic enzymes, proteases, pectases, and xylose isomerases, the domain is preferably metal-free.
The enzymatic reactions controlled by the domains of the present invention are also preferably used in enzyme-based cleaning agents, where the cleaning agent is preferably selected from the group consisting of enzyme-based laundry detergents, dishwashing detergents, grease removers, and spot removers. Also preferred are enzymatic reactions used in assays, and these assays are preferably clinical assays or industrial assays.
The method of the present invention is useful in controlling enzymatic reactions used to make products that are organic molecules selected from the group consisting of an intermediate in an organic synthesis and a final product of an organic synthesis. Also, the method of the present invention is useful in controlling enzymatic reactions used to make a biomolecule selected from the group consisting of an intermediate in a biochemical process and a final product of a biochemical process.
6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to the alpha and beta domains of metallothionein and analogs thereof, their synthesis, therapeutic applications, and methods for controlling in vitro and industrial enzymatic reactions using them. Purified metal-free and metal-containing alpha and beta domains of metallothionein and analogs thereof are provided as described below. A high yield method of synthesis and purification is also provided for the metal-free and metal containing alpha and beta domains of metallothionein and analogs thereof. Finally, methods for controlling in vitro and industrial enzymatic reactions are provided wherein the enzymatic processes are controlled by transfer of zinc to an enzyme zinc-inhibitory site or removal of zinc from an enzyme zinc inhibitory site using the alpha and beta domains of metallothionein and analogs thereof.
a. THE AND β DOMAINS OF METALLOTHIONEIN. ANALOGS
THEREOF. AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM i. THE α AND β DOMAINS OF METALLOTHIONEIN AND
ANALOGS THEREOF The present invention is directed in part to polypeptides synthesized by the procedures described below. Specifically, the polypeptides of the present invention include purified synthetic polypeptides with the same primary structure (i.e. amino acid sequence) as the alpha or beta domains of metallothionein, where these polypeptides have been synthesized according to the synthetic procedures described below. Preferably, the sequences of the polypeptides correspond to the sequences of the alpha and beta domains of the isoforms of human metallothionein, including the four known isoforms (MT-1 through 4) and those as yet unidentified. More preferably, the sequences of the polypeptides correspond to the sequences of the alpha and beta domains of human metallothionein-2 (MT-2). The primary sequence of human metallothionein-2 beta domain is:
MDPNCSCAAGDSCTCAGSCKCKECKCTSCKK
The primary sequence of human metallothionein-2 alpha domain is:
KSCCSCCPVGCAKCAQGCICKGASDKCSCCA
The present invention is also directed to analogs of the alpha or beta domains of metallothionein, where these analogs are also synthesized according to the synthetic procedures described below. The primary sequences of these analogs are related to but not identical to those of the alpha or beta domains of metallothionein. The tertiary structures of these analogs maintain similar three-dimensional arrangements of the cysteine residues to those in the alpha or beta domains of metallothionein. Thus, these analogs are capable of binding metal atoms in an analogous fashion to the metal binding of the alpha or beta domains of metallothionein.
The differences in primary sequences of the analogs as compared to the alpha or beta domains of metallothionein are
(a) substitution of individual residues with different amino acids (either naturally occurring or non-naturally occurring amino acids, including but not limited to side-chain modified amino acids),
(b) one or more repetitions of, or combinations of, the primary sequences of alpha and/or beta domains of metallothionein (with or without spacer sequences to separate the repeated or combined sequences) to provide larger polypeptides with more binding sites,
(c) one or more additional peptide residues grafted to the N-terminal and/or C-terminal end or ends of the alpha or beta domains of metallothionein, or
(d) a combination of two or more of the substitution of residues, repetition/combination of sequences, and addition of N-terminal and/or C-terminal residues described in the preceding subparagraphs (a), (b), and (c). Synthesis of the analogs is accomplished using the methods described below.
With the analogs, the change in primary structure will accomplish one or more of the following desired effects: (1) it will change the metal binding constants so as to facilitate metal release or so as to inhibit release of metals picked up through administration of the metal- free domains; (2) it will provide suitable functionality for derivatizing and/or solubilizing the domains; and (3) it will provide additional metal binding sites to increase the quantity of metal atoms released or picked up through administration of metal containing and/or metal- free domains.
Alteration of the primary structures to form analogs may also alter the cofactor binding sites of the domains. Alteration of the cofactor binding sites enhances or diminishes the effects of the cofactors on the metal uptake and release of the analogs. The domains of the present invention then can be tuned so as to optimize the metal binding to suit a particular therapeutic need.
The domains of the present invention also include derivatives, where such derivatives are domains that are bound to another atom, molecule or ion, where such other molecule or ion is either monomeric or polymeric, and where this other molecule or ion is not itself a domain.
The domains of the present invention may be metal-free or may contain one or more metal atoms.
ii. SITE SPECIFIC TARGETING In one embodiment of the present invention, a domain of the present invention is derivatized so as to be bound to an antibody or to a cellular antigen to target particular sites for release or uptake of metal ions at that site. Such derivatization is well known to those skilled in the art.
iii- DOMAINS INCLUDING PROBE CHARACTERISTICS
In one embodiment of the present invention, a domain of the present invention includes one or more probe characteristics. Non-limiting examples of probe characteristics useful in the invention are incorporation of one or more radioactive metals into a domain, isotopic substitution of one or more atoms in the domain, fusion to a domain of one or more of a fiuorophore, an NMR contrast reagent, or a Foerster radiation-less energy transfer reagent, and arsenoazo modification of amino acid residues such as tyrosine (B. L. Vallee et al., Biochem. 11, 2584, 1972.). These and other probe characteristics and methods for their incorporation into proteins are well known to those skilled in the art. b. SYNTHESIS OF THE α AND β DOMAINS OF METALLOTHIONEIN
AND ANALOGS THEREOF
Another embodiment of the present invention is a method for synthesis of the purified domains of the present invention in high yield and purity. Prior syntheses of the α and β domains of metallothionein and other cysteine-rich polypeptides have employed N-alpha-t-boc-S-(4-methylbenzyl)-L-cysteine and N-alpha-fmoc-S-benzyl-L-cysteine. These syntheses have provided the desired polypeptides in very low yield (3 percent) and only 90 percent purity.
It has now been discovered that surprisingly and unexpectedly, substituting N-alpha-finoc-S-acetimidomethyl-L-cysteine ("Fmoc-Cys(Acm)") for the N-alpha-t-boc-S - (4-methylbenzyl)-L-cysteine or N-alpha-fmoc-S-benzyl-L-cysteine permits solid-phase synthesis of the desired polypeptides in about 90 percent yield, and because of the large quantity so obtained, in as high a purity as is desired for a given purpose. Fmoc-Cys(Acm) is available commercially from Calbiochem-Novabiochem Corp. (San Diego, California). Using synthetic methods well known in the art, and using Fmoc-Cys( Acm) as the protected source of cysteine, the problems of low yield and inability to purify synthetic cysteine-rich polypeptides has been overcome. Standard procedures for deprotecting the other amino acid side groups are employed, and the removal of the Acm group is accomplished in a separate step using a Ag(I) salt (e.g. AgBF4). Thus, in this embodiment of the invention, a method is provided for synthesis and purification of a domain having a primary amino acid sequence comprising the following steps:
(a) synthesizing the domain using a solid support and at least two alpha amino acids having alpha amino groups, wherein the alpha amino acids are selected from the group consisting of amino acids with aliphatic group containing side chains wherein said aliphatic group is hydrogen or alkyl, amino acids with aromatic group containing side chains, amino acids with sulfur group containing side chains wherein said sulfur group is a thiol or a thiol ether, amino acids with hydroxyl group containing side chains, amino acids with amine group containing side chains, amino acids with guanidinium group containing side chains, amino acids with carboxylate group containing side chains, and amino acids with amide group containing side chains, wherein the alpha amino groups are protected with a protecting group selected from the group consisting of Fmoc, t-Boc, and CBZ, the carboxylate groups are protected with a protecting group selected from the group consisting of t-butyl ester and benzyl ester, the hydroxyl groups are protected with a protecting group selected from the group consisting of t-butyl ethers, and dimethylphosphate esters, the amine groups are protected with a protecting group selected from the group consisting of t-Boc and CBZ, and the thiol groups are protected with an acetimidomethyl group;
(b) cleaving the peptide synthesized in step (a) from the solid support and removing the non-acetimidomethyl protecting groups;
(c) purifying the peptide obtained from step (b); (d) precipitating the peptide obtained from step (c); and
(e) removing the acetimidomethyl protecting group with a solution comprising a silver(I) salt; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, an analog of the alpha domain of metallothionein, and an analog of the beta domain of metallothionein; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein,
(vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine,
(vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv),
(v), (vi) and (vii). Preferably, the method step (a) is accomplished using an automated solid- phase synthesizer.
Also, preferably, the domain is the alpha domain of metallothionein or the beta domain of metallothionein.
In a preferred embodiment of the method, the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t- butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group. In another preferred embodiment of the method, the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; and the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid. An example of such a gel is Sephadex 25.
In yet another preferred embodiment of the method, the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.
Most preferably, the domain synthesized according to the method is the alpha domain of metallothionein or the beta domain of metallothionein; and wherein the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t-butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group; wherein the synthesizing step (a) is accomplished using a solid-phase synthesizer wherein the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; wherein the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid; and wherein the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.
Examples of amino acids with aliphatic group containing side chains wherein said aliphatic group is hydrogen or alkyl include but are not limited to glycine (Gly, G), alamne (Ala, A), Valine (Val, V), Leucine (Leu, L), isoleucine (He, I), and proline (Pro, P). Examples of amino acids with aromatic group containing side chains include but are not limited to phenylalanine (Phe, F), tryptophan (Tip, W), and histidine (His, H). Examples of amino acids with sulfur group containing side chains wherein said sulfur group is a thiol or a thiol ether include but are not limited to cysteine (Cys, C) and methionine (Met, M). Examples of amino acids with hydroxyl group containing side chains include but are not limited to tyrosine (Tyr, Y), serine (Ser, S) and threonine (Thr, T). An example of an amino acid with an amine group containing side chain includes but is not limited to lysine (Lys, K). An example of an amino acid with a guanidinium group containing side chain includes but is not limited to arginine (Arg, R). Examples of amino acids with carboxylate group containing side chains include but are not limited to aspartic acid (Asp, D) and glutamic acid (Glu, E). Examples of amino acids with amide group containing side chains include but are not limited to asparagine (Asn, N) and glutamine (Gin, Q). One skilled in the art realizes that there are many other possible non-naturally occurring amino acids that are known and useful in the synthesis of peptides, and the method and domains of the present invention may employ these other amino acids, as well.
The method of synthesis and purification of the present invention will now be illustrated by means of a non-limiting example of the synthesis of the alpha and beta domains of human metallothionein. An ABI 433 A solid phase peptide synthesizer was loaded with Fmoc
Lys(boc)-Wang resin (p-benzyloxybenzyl resin, Bachem, Switzerland) All amino acids used in the synthesis were N-alpha-frnoc protected, and the side-chain functional groups were protected as follows: O-tert-butylaspartic acid, O-tert-butylglutamic acids, O-tert- butylserine, O-tert-butylthreonine, lysine(Boc), and Cys(Acm); all protected amino acids were obtained from Bachem. Each protected amino acid was added to the synthesizer in
5 turn, and an Fmoc/HBTU monitor was used to follow the incorporation into the peptide. On completion of the synthesis, the peptides were cleaved and the non-Acm protecting groups removed using a solution of 0.75 g. phenol, 0.25 mL ethanedithiol, 0.5 mL thioanisole, 0.5 mL water, and 10 mL trifluoroacetic acid. The peptides were precipitated with t-butyl methyl ether, and then purified by gel filtration chromatography on Sephadex
10 25 in 0.1 % trifluoroacetic acid. The Acm group was then removed by dissolving the peptide in acetic acid containing silver nitrate and stirred at 0 °C for 1 hour. The peptides were precipitated with ether, isolated by centrifuge, then treated with dithiothreitol in acetic acid at 25 °C for 3 hours. The final peptides were desalted with Sephadex 15 under a nitrogen atmosphere to yield respectively 38 mg of the alpha-domain and 60 mg of the beta-
15 domain. The purity of the peptides was determined by electrospray mass specfrometry and was higher than 93 %.
c. CONTROL OF ENZYMATIC REACTIONS USING METAL
INHIBITION
20 It has now been discovered that surprisingly, a number of metabolically critical and industrially useful enzymes are inhibited by metal cations, and in particular, by nanomolar concentrations of zinc, and that metal-free domains including thionein (T) reverses this inhibition. This inhibition is due to a metal cation binding inhibitory site that is distinct from metal sites that are part of the enzyme active sites (catalytic metal sites). The stability
25 constants for metal inhibitory sites typically are nanomolar, while those of catalytic metal sites are typically in the picomolar range. Hence such metal-inhibited enzymes cannot be identified by the criteria which were established to recognize metalloenzymes with catalytic or structural metal atoms whose biological activity is a direct function of their total metal content, but may be identified by simple assays of their activity in the presence and absence
30 of zinc and a removal agent such as a metal-free domain of the present invention.
Metal-free domains prove to be very effective agents for the removal of zinc and other metals from such inhibitory sites in metal-inhibited enzymes, and metal-containing domains release metals to provide inhibition of these enzymes. Thus, both metal-free and metal-containing domains are useful for controlling enzymatic reactions in industrial,
35 metabolic, and signal transduction pathways using site-specific metal-free and domain- reversible inhibitory metal binding to enzymes. Such control of enzymatic reactions is useful in treating pathological conditions, and is also useful in controlling in vitro, household, and industrial processes employing enzymes.
Control of enzymatic reactions for purposes of the present invention means the ability to selectively increase or decrease the rate of the reaction converting a substrate to a product, catalyzed by the enzyme. Enzyme reaction rates are expressed by those skilled in the art using a variety of terms, and these terms may depend on whether or not the reaction in question obeys particular rate laws. For purposes of this invention, the reaction rates are expressed in terms of a ratio of modified enzyme turnover rates in the presence and absence of an added domain of the present invention. The normal (unmodified) enzyme turnover number is the number of substrate molecules converted into product by an enzyme molecule in a unit of time when the enzyme is fully saturated with substrate. The modified enzyme turnover number used to express the reaction rates of the present invention is the number of substrate molecules converted into product by an enzyme molecule in one second under the particular reaction conditions employed in a process of interest. An increase in the reaction rate due to addition of a metal-free or metal-containing domain of the present invention is expressed as a ratio of the modified enzyme turnover number in the presence of added domains divided by the modified enzyme turnover number in the absence of any added domains of the present invention. This increase in reaction rate may be illustrated as follows: for a particular set of reaction conditions in the absence of added domains of the present invention, an enzyme might convert 100,000 substrate molecules into product in 1 second; when a particular amount of a domain of the present invention is added to the reaction but with the reaction conditions remaining otherwise unchanged, the same enzyme might convert 110,000 substrate molecules into product in 1 second; the increase in the reaction rate due to the added amount of domains is thus 110,000/100,000 = 1.1. A decrease in the reaction rate due to addition of a metal-free or metal-containing domain of the present invention is expressed as the ratio of the modified enzyme turnover number in the absence of added domains divided by the modified enzyme turnover number in the presence of any added domains of the present invention. This decrease in reaction rate may be illustrated as follows: for a particular set of reaction conditions in the absence of added domains of the present invention, an enzyme might convert 100,000 substrate molecules into product in 1 second; when a particular amount of a domain of the present invention is added to the reaction but with the reaction conditions remaining otherwise unchanged, the same enzyme might convert 91,000 substrate molecules into product in 1 second; the decrease in the reaction rate due to the added amount of the domains is thus 100,000/91,000 = 1.1. For purposes of the present invention, the change in reaction rate is called a factor, where the factor in the preceding examples is equal to 1.1. In one embodiment of the present invention, a method is described for controlling an enzymatic reaction susceptible to such control, the reaction comprising conversion using the enzyme of a substrate to a product; the method comprising adding an amount of a composition comprising a domain to a reaction mixture comprising an enzyme; wherein the amount is sufficient to effect a change in a rate of the reaction, wherein the change is selected from the group consisting of an increase in the rate of reaction by a factor of at least about 1.1 and a decrease in rate of reaction by a factor of at least about 1.1; wherein the enzyme comprises an inhibitory site that binds a metal cation; wherein the domain is added to the reaction mixture at a time selected from the group consisting of a time before which the substrate is available to the enzyme the and a time during which the substrate is available to the enzyme; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of (i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and
(viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii). In a preferred embodiment, the domain is the alpha domain of metallothionein or the beta domain of metallothionein. In another preferred embodiment, the domain is an analog of the alpha domain of metallothionein wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
(i) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (ii) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids. A more prefeπed embodiment uses an analog of the alpha domain of metallothionein where the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein by fusion of one primary sequence of the beta domain of metallothionein to the N-terminal end of the alpha domain of metallothionein.
The domains useful in control of enzymatic reactions are preferably purified. Purified domains comprise no more than 5 percent impurities arising from the synthesis and isolation of the domain, and more preferably, no more than 2 percent impurities arising from the synthesis and isolation of the domain. Other purified compositions useful in the enzyme control methods of the present invention use compositions where the domain comprises at least 95 % of the total protein in the composition, and preferably, where the domain comprises at least 98 % of the total protein in the composition.
As shown by way of example in Section ό.c.iii., infra, the alpha and beta domains of MT have different binding affinities for zinc. In particular, the beta domain releases zinc faster than the alpha domain. Thus, in a preferred embodiment, the alpha domain is used in embodiments wherein zinc sequestration is desired, and the beta domain is used in embodiments wherein zinc delivery is desired.
i. Control of Industrial Enzymatic Reactions
Enzyme processes are important in various industries. These industries include but are not limited to food and beverage industries such as sweetening, baking, brewing, oil and shortening, cheesemaking, distilling, meat-packing, and fruits and vegetables (particularly juice), household and industrial cleaning products industries, particularly in laundering and stain removal applications, and in the fine and commodity chemical industries.
(1) Control of Enzymatic Reactions in the Food and Beverage Industries Food industries use a great number of reactions employing enzymes susceptible to control by the domains of the present invention. Non-limiting examples of such enzymes are glucose isomerases, amylolytic enzymes, proteases, lipases, pectases, and xylose isomerases.
Commercially important glucose isomerases may be inhibited by the following metal cations: Ag(I), Ni(II), Hg(II), Fe(II), Zn(II), Ca(II), Al(III), Ga(II), Ba(II), As(III), and Cu(I, II). Such enzymes are used in the sweetener industry. Thus, in one embodiment of the present invention, a method for controlling reactions that use a glucose isomerase is described comprising adding to the reaction one or more domains of the present invention.
Commercially important amylolytic enzymes are used in sweetener industry, in brewing, in baking, and in distilleries. Alpha amylases have calcium binding sites, where Ca(II) provides conformational stability for the active site. In one embodiment of the present invention, a method for controlling reactions that use amylolytic enzymes is described comprising adding to the reaction one or more domains of the present invention. In a preferred embodiment, the domain is metal-free.
Commercially important proteases are used in meat tenderizing, texturing, beer chill-proofing, and cheese manufacturing. Subtilsins have divalent Ca(II) binding sites which serve to stabilize conformations. The action of papains and calpains is also know to be dependent on Ca(II). In one embodiment of the present invention, a method for controlling reactions that use protease enzymes is described comprising adding to the reaction one or more domains of the present invention. In a preferred embodiment, the domain is metal-free.
Commercially important lipases are used in the oil and shortening industries to hydrolyze triglyceryl esters. Some lipases require a Ca(II) cofactor which binds in the active site. In one embodiment of the present invention, a method for controlling reactions that use lipase enzymes is described comprising adding to the reaction one or more domains of the present invention. In a preferred embodiment, the domain is metal- free.
Commercially important pectases are used in the fruit and vegetable industries, particularly in the preparation of juices. Pectate lyase requires Ca(II) for activity; with pectinesterase, low concentrations of metal cations (e.g. Ca(II)) tend to enhance activity, particularly that involved in ripening. In one embodiment of the present invention, a method for controlling reactions that use pectase enzymes is described comprising adding to the reaction one or more domains of the present invention. In a preferred embodiment, the domain is metal-free.
Commercially important xylose isomerases are used in production of high-fructose corn syrup. Some xylose isomerases require Mg(II), Mn(II), Ni(II) or Co(II) for activity. In one embodiment of the present invention, a method for controlling reactions that use xylose isomerase enzymes is described comprising adding to the reaction one or more domains of the present invention. In a preferred embodiment, the domain contains a metal selected from the group consisting of Mg(II), Mn(II), Ni(II) and Co(II). In another preferred embodiment, the domain is metal-free.
(2) Control of Enzymatic Reactions in the Household and Industrial Products Industries Enzymatic reactions are important for the efficacy of certain products sold for household and industrial uses, particularly for cleaning applications. Household and industrial cleaners and detergents employ proteases, and sometimes lipases to remove protein and lipid deposits. In particular, laundry and dishwashing detergents, grease removers, and spot removers, as well as materials used to clean up oily spills, use enzymes. Thus, in one embodiment of the present invention, a method for controlling an enzymatic reaction used in a cleaning application is described comprising adding one or more domains of the present invention to a composition comprising an enzymatic cleaning agent.
(3) Control of Enzymatic Reactions in Clinical and Industrial Assays Enzymatic reactions are used as part of clinical and industrial assays. For example, many assays used to detect disease states (a non-limiting example being diabetes) and levels of biologically relevant molecules in body fluids (a non-limiting example being pregnancy tests) and industrial mixtures (non-limiting examples being foods and beverages) use enzymatic reactions. In one embodiment of the present invention, a method for controlling an enzymatic reaction used in a clinical or industrial assay is described comprising adding one or more domains of the present invention to a composition used in the assay, the assay comprising an enzymatic reaction.
(4) Control of Enzymatic Reactions in Chemical Synthesis and Biotechnological Reactions Enzymatic reactions are used as part of chemical synthesis and biotechnological transformations. In particular, immobilized enzymes and whole cells are used in small and large scale synthesis of organic molecules and for inducing expression of biologicals in biotechnological applications.
In one embodiment of the present invention, a method for controlling an enzymatic reaction used in a organic chemical synthesis is described comprising adding one or more domains of the present invention to a composition used in the synthesis. The product of the reaction is an organic molecule selected from the group consisting of an intermediate in an organic synthesis and a final product of an organic synthesis.
In another embodiment, the method of the present invention is used in a biotechnological reaction where the product is a biomolecule selected from the group consisting of an intermediate in a biochemical process and a final product of a biochemical process.
ii. EXAMPLE: CONTROL OF ENZYMATIC REACTIONS USING THIONEIN
The control of enzymatic processes will now be illustrated using thionein (T), which is a domain consisting the metal-free alpha domain of metallothionein, the N-terminal end of which has been fused to the beta domain of metallothionein.
(1) Materials and Methods Materials. Rabbit liver thionein was prepared from cadmium MT-1 (Vasak, M.,
1991, Methods Enzymol. 205, 41-44), which was a gift from Prof. G. J. Xu, Shanghai Institute of Biochemistry, and stored under liquid nitrogen (Jacob, C. Maret, W., and Vallee, B. L., 1998, Proc. Natl. Acad. Sci. USA 95, 3489-3497). To avoid metal contamination, deionized water (resistivity of ≥ 15 MΩ cm) and metal-free pipet tips (Fisher) were used throughout. In addition, adventitious metals were removed by treatment of all buffer and substrate stock solutions with 5% (v/w) Chelex (Bio-Rad) for 2 h at room temperature and subsequent filtration through Millex-GS microfilters (Millipore). Buffers and stock solutions were purged with nitrogen gas for 30 min. Enzymes were brought into 20 mM Hepes-Na+, pH 7.5 ("buffer") by passage through a PD-10 gel filtration column (Pharmacia). Ultrapure MgSO4, MgCl2, ZnCl2, ZnSO4 and KC1 were from Johnson Matthey Chemicals Ltd., England. Ac-DEVD-AMC was from Pharmingen.
Enolase. Enolase (rabbit muscle) from Sigma which contained no detectable zinc was diluted to a final concentration of 10 nM in buffer containing 2 mM MgCl2 and activity measured spectrophotometrically at 237 nm for 2 min after the reaction was started with D- 2-phosphoglyceric acid at a final concentration of 1 mM. Analogous experiments were performed with enolase (yeast) from Sigma.
Fructose 1,6-Diphosphatase. Fructose 1 ,6-diphosphatase (rabbit liver) from Sigma which contained no detectable zinc was diluted to a final concentration of 5 nM in buffer containing 1 mM MgCl2. Enzyme assays were initiated by addition of D-fructose 1,6- diphosphate to a final concentration of 100 μM, quenched after 15 min and the phosphate released titrated with the molybdenum blue method (Tashima, Y. and Yoshimura, N., 1975, J. Biochem. (Tokyo) 78, 1161-1169). This time interval is in the linear range of phosphate production.
Glyceraldehyde 3-Phosphate Dehydrogenase. Glyceraldehyde 3-phosphate dehydrogenase (rabbit muscle) from Sigma was diluted to final concentrations of 2.5 and 10 nM in buffer containing 30 mM Na^AsO^ Glyceraldehyde 3-phosphate (30 mM stock solution) and NAD+ (15 mM stock solution) were added to final concentrations of 500 and 250 μM, respectively and the reaction monitored spectrophotometrically at 340 nm for 2 min.
Aldehyde Dehydrogenase. Yeast aldehyde dehydrogenase from Sigma was diluted to a final concentration of 155 nM in buffer containing 100 mM KC1. Acetaldehyde (40 mM stock solution) and NAD+ (5 mM stock solution) were added to final concentrations of 2 and 0.5 mM, respectively and the reaction monitored spectrophotometrically at 340 nm for 2 min.
Protein Tyrosine Phosphatase. The recombinant protein tyrosine phosphatase from Calbiochem used for our studies is a truncated form of the human T-cell protein tyrosine phosphatase with an 1 lkDa deletion at the C-terminus (Cool, D. E., Tonks, N. K., Charbonneau, H, Walsh, K. A., Fischer, E. H. and Krebs, E. G., 1989, Proc. Natl. Acad. Sci. USA 86, 5257-5261). Phosphatase was diluted to a final concentration of 20 nM in buffer. An aliquot of 4-nitrophenyl phosphate was added (100 mM stock solution) to give a final concentration of 1 mM and the formation of 4-nitrophenolate was monitored
5 spectrophotometrically at 400 nm for 2 min.
Caspase-3. Recombinant human caspase-3 from Pharmingen was diluted to a final concentration of 1.7 nM in buffer. Ac-DEVD-AMC (1.12 mM stock solution) was added to a final concentration of 22.4 μM and the formation of AMC (amino-4-methylcoumarin) monitored fluorimetrically for 5 min (excitation wavelength: 380 nm, emission wavelength:
10 440 nm) with a FluoroMax-2 (Instruments S.A., Inc.) fluorimeter (Nicholson, D. W., Ali, A., Thornberry, N. A., Villancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T.- T., Yu, V. L. and Miller, D. K., 1995, Nature 376, 37-43).
Alcohol Dehydrogenase. Alcohol dehydrogenase (horse liver) from Boehringer
15 contained 4.8 zinc atoms per dimer. Alcohol dehydrogenase was diluted to a final concentration of 100 nM in buffer. Enzymatic activity was measured by following the formation of NADH spectrophotometrically at 340 nm for 2 min after adding ethanol (6 M stock solution) and NAD+ (25 mM stock solution) to final concentrations of 330 and 1 mM, respectively.
20 Carbonic Anhydrase. Carbonic anhydrase (bovine erythrocytes) from Sigma contained 0.93 zinc atom per monomer. Carbonic anhydrase was diluted to a final concentration of 250 nM in buffer. Enzymatic activity was measured spectrophotometrically at 400 nm for 2 min after adding 4-nitrophenyl acetate (20 mM stock solution in acetonitrile) to a final concentration of 0.5 mM (Henkens, R. W. and
25 Sturtevant, J. M., 1968, J. Am. Chem. Soc. 90, 2669-2676).
Glutathione Peroxidase. Glutathione peroxidase (bovine erythrocytes) from Sigma was diluted to a final concentration of 2.4 nM in buffer. Enzymatic activity was measured spectrophotometrically at 260 nm for 1 min after adding t-butyl hydroperoxide (50 mM stock solution) and glutathione (0.2 M stock solution) to final concentrations of 5 and 2
30 mM, respectively.
Glutathione Reductase. Glutathione reductase (yeast) from Sigma was diluted to a final concentration of 8 nM in buffer. Enzymatic activity was measured spectrophotometrically at 340 nm after adding NADPH (4.5 mM stock solution) and glutathione (5 mM stock solution) to final concentrations of 50 and 2 μM, respectively.
35 Zinc inhibition. Enzymes were incubated with ZnSO4 (from 10-100 μM stock solutions in water) and then assayed under initial velocity conditions. Inhibition was instantaneous in all cases. All kinetic experiments, including spectrophotometric measurements with a Cary IE UV/VIS spectrophotometer, were performed at 25 °C.
Effect of T on enzyme activities. Enzymes were preincubated with ZnSO4 for 10 min and their remaining activity determined. Zinc concentrations sufficient to inhibit at least 50% of the enzyme's original activity were chosen. The inhibited enzymes were then incubated with T for 30 min and the recovered activity determined. In a control, T lost less than 10% of its thiol content in 30 min as ascertained by titrations with 5,5'-dithiobis(2- nitrobenzoic acid) (Jacob, C, Maret, W. and Nallee, B. L., 1998. Proc. Νatl. Acad. Sci. USA 95, 3489-3494). In another set of experiments, enzymes were incubated with T first and then enzyme activities were measured 10 min after the addition of ZnSO4. The effect of T on alcohol dehydrogenase and carbonic anhydrase was investigated by measuring enzymatic activity after preincubation of the enzymes with an excess of T for 30 min.
(2) Results Zinc Inhibition of Enzymes. A number of eukaryotic, cytosolic enzymes not known to require zinc for activity are inhibited by this metal. Among these are glycolytic enzymes, a protease involved in apoptosis, and a tyrosine phosphatase. Some of these enzymes were found to be inhibited by much lower concentrations of zinc than had been reported previously (Table 1). This effective lower concentration for inhibition is due to the fact in part that we have here avoided the use of buffers and reducing agents such as dithiothreitol or 2-mercaptoethanol that are also very effective zinc-binding agents. Further, we have reduced the level of zinc contamination of buffers and substrates to below 1 ppb. Under these conditions, the IC50 values for zinc inhibition of the enzymes studied save rabbit enolase are below 250 nM. Since inhibition is in the nanomolar range, the IC50 values are a function of the enzyme concentration which on occasion had to be lowered significantly, sometimes to the limit of sensitivity of the assay. Thus these only reflect upper limits and the true inhibition constants are likely even lower. The inhibition of caspase-3 turns out to be most notable among those examined since it reflects a virtual 1:1 stoichiometric reaction: At equimolar concentrations of enzyme (1.7 nM) and zinc only 50% of the initial enzymatic activity remains (Figure 1). Again, the 5-fold higher enzyme concentration employed in a previous study clearly required a higher zinc concentration for inhibition (Perry, D. K., Smyth, M. J., Stennicke, H. R., Salvesen, G. S., Duriez, P., Poirier, G. G. and Hannun, Y. A., 1997, J. Biol. Chem. 272, 18530-18533). Table I Zinc inhibition of enzymes
Enzyme (nM) IC50 measured, nM IC50 reported, nM caspase-3 (1.7) <10 1001} fructose 1,6-diphosphatase (5) 100 3002) glyceraldehyde 3-phosphate 150 — dehydrogenase (2.5) aldehyde dehydrogenase (155) 154 — tyrosine phosphatase (20) 200 100,0003) enolase (yeast) (10)4) 250 2,5005) 1) Perry, D. K., Smyth, M. J., Stennicke, H. R., Salvesen, G. S., Duriez, P., Poirier, G. G. and Hannun, Y. A., 1997) J. Biol. Chem. 272, 18530-18533
2) Kj ; Tejwani, G. A., Pedrosa, F. O., Pontremoli, S. and Horecker, B. L., 1976, Proc. Natl. Acad. Sci. USA 73, 2692-2695
3) 15% activity remaining; Zander, N. F., Lorenzen, J. A., Cool, D. E., Tonks, N. K., Daum, G., Krebs, E. G. and Fischer, E. H., 1991, Biochemistry 30, 6964-6970
4) enolase (rabbit muscle) (10 nM): IC50 = 1.3 μM
5) Elliot, J. I. and Brewer, J. M., 1980, J. Inorg. Biochem. 12, 323-334
Since a catalytic cysteinyl residue is known to be characteristic of glyceraldehyde 3- phosphate dehydrogenase, aldehyde dehydrogenase, protein tyrosine phosphatase, and caspase-3, we have searched for other enzymes that might have potential cysteine (or selenocysteine) ligands at their active site. Unlike the above enzymes, nanomolar concentrations of zinc do not inhibit glutathione peroxidase from bovine erythrocytes (catalytic selenocysteine) or glutathione reductase from yeast (catalytic vicinal cysteines). In the presence of 1 μM zinc, glutathione peroxidase (2.4 nM) loses only 15% of its activity. The IC50 value for glutathione reductase (8 nM) is about 10 μM. Apparently, the presence of a cysteine residue at the catalytic site is not sufficient to provide an inhibitory site: the active site of enzymes with potential metal binding ligands may be designed in such a way that they are protected from inhibition by zinc or other metal ions.
Thionein-Reversible Inhibition of Enzymatic Zinc Sites. Incubation with T reactivates zinc-inhibited enzymes as illustrated for glyceraldehyde 3-phosphate dehydrogenase (Figure 2). Reactivation is a function of the concentration of zinc relative to that of T, but apparently not of the nature of the enzyme. The concentration of T required to overcome the inhibition (Figure 2) shows that on the average T sequesters about 6 to 7 zinc atoms, coπesponding to the capacity of T to bind 7 zinc atoms. T rapidly reactivates these zinc- inhibited enzymes, e.g. during the mixing time, as illustrated for glyceraldehyde 3- phosphate dehydrogenase (Figure 3).
This remarkably efficient removal of zinc from these inhibitory sites prompted us to
5 investigate the effect of T on two zinc enzymes in addition to carboxypeptidase A and alkaline phosphatase which were studied previously (Jacob, C, Maret, W. and Nallee, B. L., 1998, Proc. Νatl. Acad. Sci. USA 95, 3489-3494). Up to a 20-fold excess of T over alcohol dehydrogenase (100 nM) results in only a marginal loss of its enzymatic activity (6%). Similarly, in the presence of a 12-fold excess of T, carbonic anhydrase (250 nM) retains
10 85% of its original activity as has been described (Li, T-Y., Kraker, A. J., Shaw III, C. F. and Petering, D. H., 1980, Proc. Νatl. Acad. Sci. USA 77, 6334-6338). Thus, low concentrations of T rapidly react with zinc bound to inhibitory sites of enzymes, but, under these conditions, it cannot abstract significant amounts of zinc from the active sites of zinc metalloenzymes.
15 (3) Discussion
Thionein and metallothionein have persistently been described as unique owing to their metal content, amino acid composition and physicochemical characteristics (Nallee, B. L. and Falchuk, K. H., 1993, Physiol. Rev. 73, 79-118). T binds zinc very tightly to form zinc/sulfur clusters that do not exist in the inanimate world. Thus far T has not proven to be
20 a particularly effective chelating agent for zinc metalloenzymes and does not inhibit or remove significant amounts of zinc from the catalytic sites of zinc metalloenzymes (Jacob, C, Maret, W. and Nallee, B. L., 1998, Proc. Νatl. Acad. Sci. USA 95, 3489-3494). However, it rapidly activates enzymes which are inhibited by zinc at what we here now refer to as specific zinc inhibitory sites; these may be contrasted with zinc active sites in
25 zinc metalloenzymes. The high specificity of both nanomolar zinc inhibition and removal of zinc from these zinc inhibitory sites is commensurate with the capacity of T to bind 7 zinc atoms and suggests the possible existence of a biological regulatory system that has not been recognized thus far.
The identity and characteristic properties of zinc metalloenzymes (Vallee, B. L. and
30 Falchuk, K. H., 1993, Physiol. Rev. 73, 79-118) are well established. Enzymes which are inhibited by zinc have also been known for a long time. However, reversal of inhibition by T had not been and could not have been described previously, since T was not available in sufficient quantities for such experimentation. We now recognized thatT reverses zinc inhibition by competing for the metal at zinc inhibitory sites of enzymes (Figure 2 and 3).
35 In point of fact, zinc metalloenzymes must be protected from loss of zinc when exposed to T. T seems to distinguish between these active and inhibitory zinc sites likely on the basis of differences in their stability constants and/or their accessibility. The stability constants for zinc inhibitory sites are nanomolar, while those of catalytic or structural zinc sites are in the picomolar range. For a relatively large molecule, access to the inner coordination sphere of active site zinc atoms is limited, perhaps because such zinc atoms usually are not on the protein surface. Structural zinc atoms, while sometimes located at the surface of proteins, are fully coordinated and, hence, not readily accessible to chelating agents. Thus, while enzymes with zinc inhibitory sites do bind zinc tightly, they are not recognized readily to contain zinc, since their isolation may require conditions where zinc dissociates, and then yield an enzyme which is partially active, because a commensurate mole fraction of zinc remains bound.
Zinc is known to inhibit multiple biological processes demonstrating that its cellular concentration must be controlled carefully and kept well below the micromolar range to preserve the activity of zinc metalloenzymes and others critical to metabolism. This is accomplished in part by the cellular MT/T system. If free zinc concentrations were to rise into the micromolar range, numerous enzymatic processes could be compromised. The binding constants for zinc in T-reversible inhibitory sites of enzymes on the one hand and in catalytically active zinc metalloenzyme sites on the other seem to establish the conditions for the availability of cellular zinc, while demonstrating the need for its efficient regulation. Dissociation of zinc from zinc metalloenzymes occurs below 10"10 M while zinc inhibition becomes sigmficant above 10"8 M. The definition of this upper set-point in the nanomolar range depends on the exact magnitudes of inhibition constants.
Zinc is bound tightly to MT (KA(zn) (Zn7MT) = 3.2 x 1013 M"1), and the zinc concentration in MT accounts for approximately 50 μM zinc of the cell. The relative abundance of the cellular MT/T couple and its unique features make it an element central in the regulation of zinc. T expression is regulated by a metal response element binding factor (MTF-1) which in turn is itself activated by zinc (Radtke, F., Heuchel, R, Georgiev, O., Hergersberg, M., Gariglio, M., Dembric, Z. and Schaffner, W., 1993, EMBO J. 12, 1355- 1362). Due to its high affinity for zinc, T sequesters zinc to low nanomolar concentrations where zinc no longer inhibits enzymes. While this mechanism leads to downregulation of zinc, upregulation mechanisms include oxidative zinc release (Maret, W. and Vallee, B. L., 1998, Proc. Natl. Acad. Sci. USA 95, 3478-3482). One may wonder whether or not such a release of zinc from MT and directed to an inhibitory site could also reflect a physiological control mechanism for the regulation of enzymatic processes. iii. EXAMPLE: ZINC TRANSFER POTENTIALS OF THE ALPHA AND BETA DOMAINS OF METALLOTHIONEIN Individual zinc-reconstituted clusters in the isolated domains were characterized and compared with those in the whole molecule. The results showed that the alpha and beta clusters differ in terms of metal binding, zinc transfer, and chemical reactivity. Although each of the two clusters of MT assembles with zinc cooperatively and independently of the other domain, their properties do not add up to those of the whole molecule.
Metal binding. The chromophoric properties of the metal-thiolate complexes of MT provide a means to assess the stability of the metal-protein complex by pH titrations. The point of half-maximum absorbance of the alpha cluster is lower than that for the beta cluster, demonstrating that zinc and cadmium bind more tightly to the alpha cluster than the beta cluster (Figure 4). In a similar titration study, both human Zn7-MT-2 and cadmium MT-2 are stabilized as compared to the sum of the individual domains (Figure 5). Thus, proton titrations show the alpha cluster to be more thermodynamically stable than the beta cluster.
Zinc transfer. When 4-(2-Pyridylazo)resorcinol (PAR) is the zinc acceptor, the amount of zinc released after one hour from the alpha cluster is 2.5-fold higher than that from the beta cluster (Figure 6). While MT contains seven zinc atoms, only one of them appears to be available for transfer to zinc-depleted sorbitol dehydrogenase (apo-SDH). Exactly one of the three zinc atoms from the beta cluster is transferred to apo-SDH just as observed with the parent protein (Figure 7A). However, less than one zinc atom is transferred to apo-SDH from the alpha cluster (Figure 7B). Unlike the PAR system, the release of zinc from the beta cluster and its transfer to apo-SDH is almost ten times faster than that from the alpha cluster, suggesting different mechanisms of zinc transfer to these two acceptors. In both cases, the beta cluster releases zinc faster than the alpha cluster. Chemical reactivity. Under pseudo-first-order rate conditions when the concentration of the disulfide 5,5'-Dithiobis 2-nitrobenzoic acid (DTNB) is less than one equivalent per sulfur ([DTNB]«[cluster thiols]), the beta cluster reacts faster than the alpha cluster (Figure 8). In terms of zinc transfer and thiol reactivity with disulfides, the whole MT molecule is less reactive than its individual clusters, demonstrating that domain interactions in the whole molecule affect the reactivity of MT in addition to its effect on the structure of MT.
Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications, as would be obvious to a person skilled in the art, are intended to be included in the scope of the following claims.

Claims

WE CLAIM:
1. A method for controlling an enzymatic reaction susceptible to such control, the reaction comprising conversion using an enzyme, of a substrate, to a product; the method comprising adding an amount of a composition comprising a domain to a reaction mixture comprising an enzyme; wherein the amount is sufficient to effect a change in a rate of the reaction, wherein the change is selected from the group consisting of an increase in the rate of reaction by a factor of at least about 1.1 and a decrease in rate of reaction by a factor of at least about 1.1; wherein the enzyme comprises an inhibitory site that binds a metal cation; wherein the domain is added to the reaction mixture at a time selected from the group consisting of a time before which the substrate is available to the enzyme and a time during which the substrate is available to the enzyme; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of (i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine,
(v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids,
(ii) substitution of one or more cysteine residues with selenocysteine, (iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (v) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine, (vi) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vii) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (viii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v), (vi) and (vii).
2. The method according to claim 1, wherein the domain is the alpha domain of metallothionein or the beta domain of metallothionein.
3. The method according to claim 1, wherein the domain is an analog of the alpha domain of metallothionein wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of (i) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (ii) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids.
4. The method according to claim 3, wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein by fusion of one primary sequence of the beta domain of metallothionein to the N-terminal end of the alpha domain of metallothionein.
5. The method according to claim 1, wherein the domain comprises no more than 5 percent impurities arising from the synthesis and isolation of the domain, and
6. The method according to claim 1, wherein the domain comprises no more than 2 percent impurities arising from the synthesis and isolation of the domain.
7. The method according to claim 1, wherein the domain comprises at least 95 % of the total protein in the composition.
10 8. The method according to claim 1, wherein the domain comprises at least 98 % of the total protein in the composition.
9. The method according to claim 1, wherein the domain is metal containing.
15 10. The method according to claim 9, wherein the domain contains one or more metals selected from the group consisting of main group metals, transition metals, lanthanides, and actinides.
11. The method according to claim 9, wherein the domain contains one or more metals 20 selected from the group consisting of zinc, copper, gold, cadmium, iron, cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technetium, lanthanum, and combinations thereof.
25 12. The method according to claim 9, wherein the metal is zinc.
13. The method according to claim 1 , wherein the domain is metal-free.
14. The method according to claim 4, wherein the domain is metal-free.
30
15. The method according to claim 1 wherein the enzyme is selected from the group consisting of glucose isomerases, amylolytic enzymes, proteases, lipases, pectases, and xylose isomerases.
35 16. The method according to claim 15 wherein the enzyme is a glucose isomerase and wherein the domain contains at least one metal selected from the group consisting of Ag(I), Ni(II), Hg(II), Fe(II), Zn(II), Ca(II),Al(III), Ga(II), Ba(II), As(III), Cu(I, II) and combinations of these metals.
17. The method according to claim 15 wherein the enzyme is an amylolytic enzyme and 5 wherein the domain is metal-free.
18. The method according to claim 15 wherein the enzyme is a protease and wherein the domain is metal-free.
10 19. The method according to claim 15 wherein the enzyme is a lipase and wherein the domain is metal-free.
20. The method according to claim 15 wherein the enzyme is a pectase and wherein the domain is metal-free.
15
21. The method according to claim 15 wherein the enzyme is a xylose isomerase and wherein the domain contains at least one metal selected from the group consisting of Mg(II), Mn(II), Ni(II), Co(II), and combinations of these metals.
20 22. The method according to claim 15 wherein the enzyme is a xylose isomerase and wherein the domain is metal free.
23. The method according to claim 1 wherein the reaction mixture comprises a cleaning agent.
25
24. The method according to claim 23 wherein the cleaning agent is selected from the group consisting of laundry detergents, dishwashing detergents, grease removers, and spot removers.
30 25. The method according to claim 1 wherein the reaction mixture comprises an assay.
26. The method according to claim 25 wherein the assay is selected from the group consisting of clinical assays and industrial assays.
35 27. The method according to claim 1 wherein the product is an organic molecule selected from the group consisting of an intermediate in an organic synthesis and a final product of an organic synthesis.
28. The method according to claim 1 wherein the product is a biomolecule selected from the group consisting of an intermediate in biochemical process and a final product of a biochemical process.
29. The method according to claim 1 wherein the inhibitory site binds zinc.
30. The method according to claim 1 wherein the enzyme is immobilized.
31. The method according to claim 29 wherein the enzyme is immobilized as part of a whole cell.
AMENDED CLAIMS
[received by the International Bureau on 17 July 2000 (17.07.00) original claim 1 amended; remaining claims unchanged (1 pages)]
1. A method for controlling an enzymatic reaction susceptible to such control, the reaction comprising conversion using an enzyme, of a substrate, to a product; the method comprising adding an amount of a composition comprising a domain to a reaction mixture comprising an enzyme; wherein the amount is sufficient to effect a change in a rate of the reaction, wherein the change is selected from the group consisting of an increase in the rate of reaction by a factor of at least about 1.1 and a decrease in rate of reaction by a factor of at least about 1.1; wherein the enzyme comprises an inhibitory site that binds a metal cation; wherein the enzyme is not carboxypeptidase A, wherein the domain is added to the reaction mixture at a time selected from the group consisting of a time before which the substrate is available to the enzyme and a time during which the substrate is available to the enzyme; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of
(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids,
(ii) substitution of one or more cysteine residues with selenocysteine,
(iii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains are optionally substituted with selenocysteine,
(iv) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, and wherein one or more cysteine residues of the alpha domains is optionally substituted with selenocysteine, and wherein one or more cysteine residues of the beta domains is optionally substituted with selenocysteine,
(v) one or more primary sequences of the beta domain of metallothionein
PCT/US2000/004734 1999-02-26 2000-02-25 A method for controlling an enzymatic reaction by the alpha and beta domains of metallothionein WO2000050448A1 (en)

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CODY ET AL.: "Replacement of All alpha-Domain Lysines with Glutamates Reduces Metallothionein Detoxification Function", BIOCHEMICAL & BIOPHYSICAL RESEARCH COMMUNICATIONS,, vol. 202, no. 2, 29 July 1994 (1994-07-29), pages 954 - 959, XP002927897 *
JACOB ET AL.: "Control of Zinc Transfer Between Thionein, metallothionein and Zinc Proteins", PROC. NATL. ACAD. SCI. USA,, vol. 95, no. 7, 31 March 1998 (1998-03-31), pages 3489 - 3494, XP002927895 *
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PAN ET AL.: "Substitution of Glutamic Acids for the Conserved Lysines in the alpha Domain Affects Metal Binding in Both the alpha and beta Domains of Mammalian Metallothionein", BIOCHEMICAL & BIOPHYSICAL RESEARCH COMMUNICATIONS,, vol. 202, no. 1, 15 July 1994 (1994-07-15), pages 621 - 628, XP002927898 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2813529A1 (en) * 2000-09-06 2002-03-08 Provital S A Topical compositions, used to protect against heavy metals and ultraviolet radiation, contain metallothionein proteins
ES2198171A1 (en) * 2000-09-06 2004-01-16 Provital S A Topical compositions, used to protect against heavy metals and ultraviolet radiation, contain metallothionein proteins
US8258181B2 (en) 2005-03-23 2012-09-04 Florida Atlantic University Treatment or prevention of cancer and precancerous disorders
US8357720B2 (en) 2005-03-23 2013-01-22 Florida Atlantic University Treatment or prevention of cancer and precancerous disorders
US8603985B2 (en) 2005-03-23 2013-12-10 CHS Pharma, Inc Treatment or prevention of cancer and precancerous disorders
US8765808B2 (en) 2005-03-23 2014-07-01 Chs Pharma, Inc. Treatment or prevention of cancer and precancerous disorders
WO2007130575A2 (en) * 2006-05-03 2007-11-15 Florida Atlantic University Protection against oxidative damage in cells
WO2007130575A3 (en) * 2006-05-03 2008-10-02 Univ Florida Atlantic Protection against oxidative damage in cells
CN107384996A (en) * 2017-06-26 2017-11-24 盘古基因生物工程(南京)股份有限公司 Method for inducing saccharomyces cerevisiae to produce metallothionein with high yield and application of method
CN107384996B (en) * 2017-06-26 2020-10-23 盘古基因生物工程(南京)股份有限公司 Method for inducing saccharomyces cerevisiae to produce metallothionein with high yield and application of method
CN110153440A (en) * 2019-05-14 2019-08-23 桂林理工大学 A kind of aspergillus japonicus fermentation liquid green prepares the method and application of nano silver

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