TWI362417B - Crystal structure qc 8.0 of soluble glutaminyl cyclase - Google Patents

Description

[Technical Field] The present invention relates to a crystal structure, and more particularly to the crystal structure of a glutamine amine cyclase. [Prior Art] The formation of N-terminal luciferin (N_terminal PGlu) is an important post-translation or co-translational modification during the process of sequestration of several biologically active neuropeptides, hormones and cytokine secretion pathways. In forming the proper configuration of these regulatory peptides, it is desirable that the N-terminal pyrophyllin binds to its receptor and/or protects the n-terminus of these peptides from the decomposition of exopepetidase (Van Coillie et al.,

Biochemistry 37: 12672-12680 (1998); Hinke et al, J. Biol. Chem. 275: 3827-3834 (2000)) 〇N-terminal pyroglutamic acid is a coating of its glutamine-based precursors. form. Glutaminyl CyClaSe (QC) is responsible for the subsequent translational modification of the catalyst (Fischer et al., Proc. Natl. Acad.

Sci. USA 84: 3628_3632 (1987); Busby et al., J. Biol. Chem. 262: 8532-8536 (1987)). QC (EC 2.3.2.5) is an acyltransferase that is derived from animals or from plants (Fischer et al., proc. Natl. Acad. Sci. USA 84: 3628-3632 (1987); Busby et al. J. Biol. Chem. 262: 8532-8536 (1987); Oberg et al., Eur. J. Biochem. 258:214-222 (1998)). High amounts of qC can be found in neuroendocrine tissues of mammals, such as hypothalamics and pituitary (Busby et al, j Bi〇1 chem 3 1362417 262: 8532-8536 ( 1987); Sykes et al., FEBS Lett. 455: 159-161 (1999)). Whether it is from yeast or even from human qc, its amino acid sequence is highly conserved. Although studies have shown that QCs from animals are similar in molecular weight to plant-derived qc, ie between 33 and 40 kDa, QCs from animals have been found to have significantly different structural and protein stability from plant-derived qc (Oberg et al. Eur. J. Biochem. 258: 214-222 (1998); Schilling et al, Biochemistry 41: 10849-10857 (2002)). Although there have been no reports of bacterial QCS as of now, QCs from mammals have been predicted to have a high degree of similarity to the bacterial doublei-zinc aminopeptidase (Schilling et al., J Biol. chem. 278: 49773-49779 (2003); Bootii et al., BMC biol. 2: 2 (2004)) Several human genetic diseases, such as osteoporosis of multi-inherent hormonal lesions, characterized by Bone mass reduction and microstructural degradation of bone tissue (Stewart et al., J. Endocrinol. 166: 235-245 (2000)), shown by the variation of the QC gene. The QC coding gene (QPCT) is located on chromosome 2p22 .3, in this region, the analysis of 13 single nucleotide polymorphisms has been shown to be significantly associated with osteoporosis susceptibility in adult women (Ezura et al., J). Bone Miner. Res. 19: 1296-1301 (2004X). Among these single nucleotide polymorphisms, R54W is statistically the most obvious relationship with osteoporosis, and 4 1362417 shows its line through the lower view. mound - The pituitary gland-gonadal central system affects the onset (Ezura et al., J. Bone Miner. Res. 19: 1296-1301 (2004)). Interestingly, 'QC also catalyzes the cyclization of N-terminal pyroglutamic acid , leading to the formation of pyroglutamic acid (Schilling et al, FEBS Lett. 563: 191-196 (2004)). This reaction may be related to the formation of several plaque-forming peptides, for example, Alzheimer's disease plays an important role in amyloid-β peptide (Αβ peptide), collagen protein Azheimer's amyloid • c〇llagen_like Alzheimer amyloid plaque component (CLAC) (Morgan et al., pr〇g. Neurobiol. 74: 323-349 (2004); Hashimoto et al., EMBO J. 21: 1524-1534 (2002)). A peptide containing N-terminal pyroglutamic acid, for example The pGlu3-Ap peptide is the major component of the amyloid beta peptide in the nucleus of neuroinflammatory plaques (Said〇 et al., Neuron 14: 457-466 (1995); Kuo et al., Bi 〇chem. Biophys. Res. Commun. 237. 188-191 (1997); Russo et al., j. Neurochem. 82: 1480-1489 ® (2002)). N-terminal pyroglutamic acid increases the hydrophobicity, protein breakdown, enthalpy and neurotropism of these peptides (Russo et al, j. Neurochem 82: 1480-1489 (2002); Harigaya Biochem. Biophys. Res. Commun. 276: 422-427 (2_), which may cause excessive accumulation of pyro-facial acid-late a powdery beta peptide in several senile plaques (10), thereby accelerating the development of neurodegenerative diseases. So far, there are several different theoretical assumptions about the nature and structure of Qc from humans and animals. The present invention provides a free form of the Qc crystal structure, the structure of the active site or catalytic core of QC, a method for identifying an inhibitor of qc, and a structural basis for rationally designing an inhibitor against a disease associated with qC. SUMMARY OF THE INVENTION One aspect of the present invention provides a crystal structure of glutaminyl cyclase (QC). Another embodiment of the present invention provides a crystal composition comprising a complex comprising at least a -QC molecule and another molecule that interacts or binds to the Qc molecule. Another aspect of this month provides a method for identifying inhibitors of glutamate cyclase (QC). The method comprises the steps of: (8) preparing a QC protein, preferably a polypeptide having an amino acid sequence of 33 to 36 amino acid residues in SEQ ID NO: 1, wherein the blister has a four-sided sequence and a sequence identifier of The active sites of the 159th, 202th, and 330th 2S夂 residues and the zinc ions coordinated by the water molecules; (8) the polypeptide: the test is to be determined to form a QC side lysate; (6) the new step (8) The three purchases of the composite, the injection of the inhibitor with the zinc ion, and the inhibition of the inhibitor of the invention are recognized as W. [Embodiment] In order to make the content of the present invention clear and easy to understand, - (10) There are noun definitions such as 1362417. Active site-dictionary refers to a characteristic region (or atom) in which a molecule (4) can interact with another molecule into a stable parent. The term "specific" in this context also refers to the reaction portion of a macromolecule that is directly involved in the other molecule and specifically binds to another molecule. In other embodiments the 'binding site may comprise or be defined as a three dimensional arrangement of one or more amino acid residues within the folded polypeptide. "Analog" '-- refers to a drug or chemical compound whose structure is related to another drug • a secret compound in a certain aspect' but whose chemical and biological properties may be similar or different. Coordinates or "structural coordinates" It refers to the Cartesian coordinates derived from the mathematical formula related to its type obtained by diffraction of a monochromatic X-ray beam of a crystalline protein or protein complex atom. The diffraction data is used to calculate the electron density map of the repeating unit of the crystal, and then the electron density map is used to establish the position of the individual atoms of the molecular or molecular complex. • “—Like refers to protein f, polypeptide, _form or part thereof, having the same amino acid sequence as the sequence identification number! QC; 'any of the functions of the county (4) secret or combined egg (4) or Structural position, wherein the sequence identifier is a human octane-base acid sequence. % 质" - any molecule to which the semaphore acts, according to the invention, the matrix binds to the active site of QC to form a qc_matrix _Complex. 7 1362417: "mature location" - refers to a portion or a fragment comprising a QC protein or analog comprising an active or catalytic site; that is, having 33 to 361 amino acid residues in SEQ ID NO: Amino acid sequence polypeptide. "root mean square error" __ The square root of the arithmetic mean of the mean squared error squared, is a way of expressing the error or variation of a trend or target. A variant of the sequence identification number 1 "polypeptide sequence" , the term includes one or more amino acid substitutions, variations, modifications, substitutions, deletions or additions, whether or not taken from a sequence that provides a final polypeptide sequence that provides a QC-active protein. The disclosure of the present invention will use the following abbreviations of amino acids, A = Ala = Alamne, T = Thr = Threonine, V = Val = Valine, C = Cys = Cysteine, L=Leu=Leucine, Y=Tyr=Tyrosine, I=Ile=Isoleucine, N=Asn=Aspartame (Asparagine), P=Pro=Proline, Q=Gln=glutamine, F=Phe=Phenylalanine, D=Asp=aspartic acid (Aspartic) Acid), W=Trp=Tryptophan, E=Glu=Glutamic acid, M=Met=Methionine, K=Lys=Lysine, G = Gly = Glycine, R = Arg = Arginine, S = Ser = Serine, H = His = Histidine. A. Replication, Expression and Purification The nucleotide sequence encoding QC or a functional fragment or derivative thereof can be inserted into a expression vector suitable for 1362417. It contains the elements required to transcribe and translate the inserted protein code sequence, and then the conventional method is used to deliver the vector into the desired host cell. For details on the methods of colonization, performance, and purification of QC, please refer to the contents of Patent Application No. 11/331,704. B. Crystal structure X-ray structure coordinates the wire in the middle point _ special configuration, well known for this technology

How easily understand the protein or enzyme/matrix complex—the group structure coordinates are the points at which the group defines the three-dimensional configuration. As long as the sum of the atomic planes and the angles maintain the necessity of marriage, it can be defined by completely different, _ coordinates, similar or identical.

The three-dimensional data can be generated by a single-instruction or a group of instructions, such as a computer program or instruction that produces a three-dimensional structure or structural image representation. Image representations can be generated or presented by commercially available software programs such as SOLVE, RESOLVE (Terwilliger et al., Methods Enzymol. 374: 22-37 (2003)) Ο (Jones et al., Acta Crystallogr. A47: 110) -119 (1991)), PROCHECK (Laskowski et al., J. Appl. Crystallogr. 26: 283-291 (1993)), MOLSCRIPT (Kraulis et al., J. appl. crystallogr. 24: 946-950 (1991)) Raster 3D (Merrit & Bacon et al., Methods Enzymol. 277: 505-524 (1997)) and GRASP (Nicholls et al., Proteins 11: 281-296 (1991)), which are incorporated herein by reference. The present invention provides a crystal structure of a QC polypeptide comprising a QC protein which is preferably a polypeptide having an amino acid sequence of the s3 to S61 amino acid residues in the sequence. One embodiment of the present invention provides a crystal composition of 〇 c obtained from a mammal. Another embodiment of the present invention provides a crystal structure obtained from a human. The present invention further provides that the crystal structure of human Qc' includes a three-dimensional structure characterized by the atomic reduction coordinates of Figure 1, and is designated "QC65". Another embodiment according to the present invention provides a crystal structure of a human QC having a space group of R32 to form a unit lattice having a size of a = b = 119.03 angstroms (4), c = 332.94 Å. A further embodiment of the invention provides a crystal having the above characteristics, wherein the crystal is diffracted to determine the atomic coordinates of the crystal at a resolution of about 1.66 humans. The present invention further provides the crystal structure of human QC, including the three-dimensional structure characterized by the atomic structure coordinates of Fig. 2, and is named "QC8". Another embodiment of the present invention provides a crystal structure for providing human QC having a space group of R32 to form a unit lattice having a size of a = b = 118.99 A and c = 332.26 A. Yet another embodiment of the present invention provides a crystal having the above characteristics in which the crystal is diffracted to determine the atomic coordinates of the crystal at a resolution of about 2.35 A. The present invention provides a Qc crystal comprising two QC molecules. According to a further embodiment, the invention provides a crystal having the above features, wherein all Ca atoms between the two QC molecules have a root mean square error of about 368.

The thermogenic region of human QC (33 to 361 amino acid residues in SEQ ID NO: 1) shows a hypothetical physiological matrix for human QC (IV) physiobgW 1362417 substrate) with face acid amine and face acid cyclase active crystal The asymmetric unit contains two human QC molecules with a == variance (with C° atom) between each other. 368 This spherical structure shows a size of 63 χ % of aa/β. The α-helix and the β-plane respectively have an amine-based It residue' and have a 6〇/〇-amino acid residue in the 3Ι0-helix region. The third figure shows a complete picture of the human QC structure, in which the six β chains in the center are orange, and the alpha helices at the top and bottom of the top are cyan, magenta and yellow, respectively. The ionic ion is displayed in a yellow sphere, and the residue of the ligated position, Arg54 (arginine 54) and sulfate ion are depicted by the ball. The crimp and loop portions adjacent to the catalytic center are green, while the portions not associated with the active site are gray, and the gray dots represent the irregular regions of the 183th to 188th amino acid residues of the sequence identifier. Fig. 3B is a diagram showing the topology of the human Qc structure' wherein the color code of the secondary structural element is the same as that of Fig. 3A.

Referring to Figures 3A and 3B, the structure has an open sandwich topology comprising two alpha helices 7 and 〇 19) and opposite six additional alpha helices (α2, α3, α4, 55, (χ6 and (Χ10) are surrounded by the central six-strand ρ plane, and are located on the side of the other two α-helices (αΐ and α8) on the side of the ρ plane (the third figure). The twisted β-plane is composed of two The reverse chain (βΐ and β2) and the four isotropic chains φ3, β4, β5 and ρ6) form the hydrophobic core of the molecule. The crimp and ring regions of the structure represent 42% of all residues. Half of them are the main components of the active site (Fig. 3B). The structure at pH 6.5 is substantially similar to pH 8.0, while the structure at pH 11 1362417 8.0 has a root mean square error between QC molecules (all c atoms). 〇A. C.QC/QC Matrix Composites Another aspect of the invention provides a composite crystal comprising QC and a Qc matrix bonded to QC. In the embodiment the crystal of the 'QC/QC matrix composite includes (a) a polypeptide having an amino acid sequence of 33 to 361 residues of SEQ ID NO: 1, or a homologue thereof; analog or variant thereof, (b) a Qc matrix such that the crystal can be X-ray diffraction two to determine the QC/QC matrix complex at a rider of approximately 2 22 A(10) sub-coordinates. The invention (IV)-embodiments provide for the inclusion of atomic structure coordinates according to Figure 4 A three-dimensional structure of QC/QC matrix composite. A further embodiment of the present invention provides a space group having R32 to form a qc/qc ray complex of a unit lattice having a size of a=b=119.14 A, e=332.61 A The 3D image of the QC composite crystal can also be generated by a single or a set of instructions, such as a computer program or instruction that produces a three-dimensional structure or structural image representation. The software can be obtained or rendered on the market. For example, the software program described in the method for determining the structure of the QC aa structure. D. Identifying the inhibitor of QC in the use of QC, the structural coordinates generated by its homologue or one of its active sites, 'mouth construction 'Time' needs to convert the structural coordinates into a three-dimensional shape or obtain three-dimensional structural information therefrom. It is well known to those skilled in the art that it can be used by a set of structures 12 1362417 : ΓΓ 2 part of the three-dimensional structure or three-dimensional image representation of the market Upper A method for obtaining a softener that can be obtained by m. For a method for identifying an inhibitor of sinking, comprising the following steps: Preparing an amine soil column I having a 33 to 361 amino acid residue in the sequence identification number! The reading of 'the towel' has the active site of the 159, 202, 33G amino acid residues in the identification number 1 of the four sides and the zinc ions coordinated by the water molecules;

(9) making the New Zealand and the female sputum into a Qc_inhibition complex; (6) generating a three-dimensional model of the QC-inhibitor complex obtained by the step (b); wherein the imidazole nitrogen is combined with zinc ions. The inhibitor to be tested is identified as an inhibitor of QC. The active site may further comprise the second, 1, 2, 7, 24, 305, 325 and 329 amino acid residues in the sequence identifier. According to an embodiment of the present invention, the active site of the QC includes a water molecule, a four-sided surface, and the 159th, 202, and 330 amino acid residues in the sequence identification number 1, and the 160th amino acid residue in the sequence identification number 1. The ligated ion, the 160th amino acid residue in the sequence number 1 forms a peptide bond with the 159th amino acid residue in SEQ ID NO: 1. In another embodiment, the peptide bond is stabilized by a plurality of hydrogen bonds and has a cis configuration. In still another embodiment, the active site of QC comprises a hydrophobic pocket arranged by 144, 146, 154, 249, 303, 321, 325 and 329 amino acid residues 13 of SEQ ID NO: 1. . Further, the active site further includes a sulfate ion adjacent to the hydrophobic bag, wherein the sulfate ion is hydrogen-bonded to the 144th, 206th, 207th, 330th amino acid residue of the sequence number 1 and at least two moisture carriers. The three-dimensional data of the QC/test inhibitor complex can also be generated by a single or a group of such programs, such as computer programs or instructions that produce a two-dimensional structure or structural image representation. A software program can be obtained or presented on the market, such as the software program described in the method for determining the QC crystal structure and the QC/QC matrix composite structure. Inhibitors include, but are not limited to, imidazole-derived inhibitors such as vinyl-l-benzylimidazole, n-benzylimidaz〇le, and N-Ν-ω-acetylhistamine For example, 'eleCtr〇n-rich niideophile, which has good binding ability to zinc ions of human Qc and binds to large hydrophobic substituents, may also be the structural basis of effective Qc inhibitors. As shown in Figure 5D, the combination of inhibitors results in the removal of six water molecules in the active site pocket, including water molecules coordinated by the imidazole nitrogen of the inhibitor.

The Qc structure coordinates or the three-dimensional image representation produced by the coordinates can be used for various purposes in conjunction with a computer, including the identification of QC inhibitors. It is also possible to use different arithmetic methods to determine whether the three-dimensional structure of the molecule or its active site is identical to all or part of its active group or its active group. Those skilled in the art can implement this method using the currently existing software application 1362417 program. E. Method of Making Face Acid Amine Cyclase Enzyme Crystals The present invention provides a method of making human QC crystals. In accordance with an embodiment of the present invention, a method of making a human QC crystal includes (expressing a QC protein; (b) purifying a QC protein; and (C) crystallizing a QC protein, to form a human QC crystal. The QC protein is preferably crystallized by a ftangmg-drop vapor diffusion method.

F. Screening for Drugs Once the possible matrices are identified, they can be selected from a pool of compounds available on the market from most large chemical companies, or they can be synthesized (symhesiz^ novo). When a suitable drug is identified, a composite crystal (C0-Crystal) can be produced, including a complex formed by QC Ba|| and a drug, which is subjected to X-ray diffraction to determine the crystal of the QC/drug complex. structure.

The present invention also encompasses a thorough matrix, which is the specific axis method described herein for the treatment of mammals (|New Zealand). The present invention will be made by the following examples, but should not be construed as limiting the scope of the invention. x Example 1: Human QC Expression and Purification The human QC cDNA was amplified by polymerase chain reaction (PCR) from the Human Bone Digital cDNA Library (Clontech, Mountain View, CA), using the previous application in Taiwan Patent Application 1362417. Several modified expression-like expression vectors (Novagen, Darmstadt, Germany, Germany) described in G94132349 express mature enzymes (residues 33-361) in E. coli cells. Eggs labeled with selenosine (SeMe soup ded) were produced in E. coli using a non-auxotrophic protoc〇i procedure. And (4) purification of the raw protein f marriage. In addition, human qc variants were constructed using the Quick Conversion Locus Point Mutagenesis Kit (Quickchange, liona mutagenesis kit) (Stratagene, La Jolla, Calif.) and expressed and purified in the same manner as wild-type human QC. Example 2: Crystals of human QC Purified human QC was concentrated to 8-10 mg/ml (mg/mL) and crystallized by suspension diffusion at 250c. Production of wild-type, labeled mela-sulphate and mutations using an equal amount of protein solution and a reservoir containing 丨6_丨8 M sulphate, 4% dioxanthon and 1 mM mM MES buffer at pH 6.5 A diamond-shaped six-sided crystal of human type qc. In the case of pH 8.0, the MES buffer in the reservoir was replaced by Tris-HCl. In the example of the combined matrix form, the crystal of the E201Q variant (growth at pH 7.) is immersed in a solution containing 75% mother liquor, 25% glycerol and U glutamic acid citrate tert-butyl nitrate for 1.5 hours, as shown in the table. The different synchrotron beamlines listed in 1 were subjected to X-ray diffraction experiments. Before the crystal was embedded in the X-ray machine, it was temporarily immersed in 20-25% glycerol (v/v) as a cryoprotectant 16 1362417. The mother liquor, using the HKL kit (〇twinowski et al., Methods Enzymol. 276: 307-326 (1997)), treats and proportions all the diffraction data, the space group of these crystals is R32, with a=b=119A, c = 333 A specific unit lattice, where the asymmetric unit comprises two human QC molecules. -$ L $ Heavy Wavelength Abnormal Diffraction

Data set Wavelength (A) Space group Resolution (A) Total observation 値 Unique reflection 値

SeMet-QC λΐ 0.9792 SeMet-QC λ2 SeMet-QC λ3 0.9794 0.9750 R32 50-1.8 (1.86-1.80)1 497051 497136 84209 84238 5.9 (5.6) 100.0(100.0) 25.2 (4.9) 27.4 (4.9) 6.4 (34.1) 6.2 (33.2) 497057 Average repetition (redundancy) Completeness (%) "Liu 21.1(4.7) Rmerge (%) 7.8 (36.0) Quality and Z値(SOLVE) 0.65 84184 丨e ) Secret __0.65,112.2 (in the analysis Degree range 15-2.0 A) 53⁄4 (KEK洸factory_tonfactory) ( Tsukuba, _like light t-bracket number 値 corresponds to the highest resolution shell 値 (resoluti〇n shdl). Example 3: human QC crystal structure Decide and correct the structure of human qc at pH 6.5 using the program SOLVE (Terwilliger et al., Methods Enzymol. 374: 22-37 (2003)) using the Muitiwaveiength anomalous diffraction phasing method. 0.9792 A (peak region) '0.9794 A (side area) and 〇975 〇 person (high energy remote area) (refer to Table 1) collected multi-wavelength anomalous diffraction data with resolution ranging from 2〇 to 2〇Α 'Finding all 14 helium atom positions successfully in the asymmetry unit' Lines of code RESOLVE (Terwilliger et al., Methods 17 1362417 Kawasaki said secret suppression ⑽3)). The cap is modified by the dissolution technique (10) to flatten the initial electron density, and the entire multi-wavelength anomalous diffraction data with a resolution of % to 自动8 is used to automatically establish a protein model of 83% or more, using the program 0 (: 丨Gnes et al. Human, Acta Crystalbgr. A 47: 11〇_119 (1991)) Control wild type crystals in the dragon group with resolution L66 A, hand other models and further correction. The modified model is used to phase the variant, the different pH values, and the isomorphic structure of the binding matrix and the binding inhibitor form. For each structure, the model establishment of the repetitive cycle by the procedure is completed and the calculation is performed by the crystallography NMR system (Bmnger et al., Acta Crystallogr. D 54: 905-921 (1998)). The Rfree value is calculated using 5% reflection. The program PR0CHECK is used to check the corrected stoichiometric quality of the structure (Lask〇wski et al., J. Appl. Crystallogr. 26: 283-291 (1993)). Each final refined structure includes 323 of the 329 residues of the human QC molecule; the 183th to 188th residues are irregular regions. Very regular water molecules are located and incorporated into the model. For example, MOLSCRIPT (Kraulis et al., J. appl. crystallogr. 24: 946-950 (1991)), Raster 3D (Merrit & Bacon et al., Methods Enzymol. 277: 505-524 (1997)) and GRASP (Nicholls et al.). The program of Humans, Proteins 11: 281-296 (1991)) produces molecular patterns. Example 4: Active site structure of human QC The active site is mainly formed by six loops between α3-α4, β3-α5, β4-β7, β5_(χ8, α8-α9&β6-α10 1362417 (Fig. 3B) The catalytic pocket is adjacent to the carboxy terminal side of the central parallel chain p3, β4, and β5 (Fig. 3A). It is rather narrow but can pass through the solvent channel to the solvent body. The single chemical ion of human QC is located at the active site. The bottom of the bag is surrounded by oxygen at the delta 2 position of aspartic acid residue 159 (D159 0δ2), oxygen at the epsilon 1 position of glutamic acid 202 (E202 Οεΐ), and nitrogen at the epsilon 1 position of histidine 330 ( Η330 Νε2) and one water molecule coordination. In addition, 'other fully conserved fesidues, including E201, W207, D248, D305, F325 and W329 (Fig. 3C) in the immediate vicinity of the zinc environment, suggest that they are The role of catalysis. As shown in Table 2, mutations in these amino acids significantly reduce enzyme activity. Acidic residues E2〇1, D248 and D305 oppose each other at pH 6.5 and 8.0, possibly between each other. Hydrogen bond formation. D159 with the word position in the subsequent S1 The peptide bond between 60 is in the cis configuration and consists of ϋ159〇δ1-Η140Νε2 (2.70 A), 81600γ-Ε2480δ1 (2 66 persons), 01590-water (2.65 persons) and 8160 team water (2.80 persons) The hydrogen bond network is stable. Table 2. Kinetic energy parameters of wild-type and mutant human qc

pH 7.5 pH 8.0 pH 8.5 pH 8.8 090 Soil 0.09 9.76 ± 1.47 11.104 ±2.716 0.63 + 0.01 8.63 ±0.48 13.663 + 0.497 0.90 ±0.05 9.93 ±0.30 11.044 ±0.331 2.06 + 0.62 8.56 ± 1.55 4.319 + 0.544

R54W K144A FI 46 A E201D Ε201(^ W207L 0.76 ± 0.04 1.47 ±〇.〇2 0.82 ±0.16 12.62 ±2.98 1.77 ±〇.〇7 7.35 ±0.26 11.67 ±0,34 7.91 ±2.14 0.87 ±0.28 0.43 + 0.01

9.704 ± 0.824 7.944 Soil 0.368 9.536 ± 0.769 0.068 Soil 0.007 ND 0.243 + 0.002 19 1362417 W207F 0.59 ±0.05 2_32 Soil 0.07 3.943 + 0 189 D248A: No' - Q304L 1.16 ±0.09 9.39 ± 1.18 8.028 + 0.386 D305Li ND _ F325A 4.67 0·24 12.91 ±0.06 2.772 + 0 132 W329A 29.53 ±2.29 1.35 ±0.07 0·046 0:001 • The number of words represented by the average ίίΐϊΐ quasi-error = 2 0Γ 3)- t is detected at pH 8.0. And D3〇5L has about 0.001% of wild-type enzyme, about 〇1% and about 0.03, respectively. ND: The hydrophobic bag of the active site cannot be measured by the residues Κ144, F146, F154, L249, 1303, 1321, F325 and W329. It has a size of about 13\11乂7 people3 and has six water molecules. Located in the bag, including water that is coordinated with zinc ions. Further, the sulfate ion is located near the mouth of the bag, and is hydrogen-bonded to Κ144Νζ, Η206ΝδBu W207N, Η330Νδ1, and several water molecules (Fig. 3C). The residues of the active site are shown and marked. The possible hydrogen bonds and coordination bonds are represented by cyan and yellow dashed lines, respectively. The green dotted line shows the possible non-D305 and E201 between sequence identification number 1 and between D305 and D248. Frequent hydrogen bonds. Example 5: Structure Preparation of Enzyme-Inhibitor Complex In a crystal form of a ligation inhibitor, a 1.5 μl protein solution containing human qC was mixed with a 〇5 μl inhibitor solution (1 mM) in a 2 μm container. The resulting crystals were subjected to a xenon ray diffraction method as described in Example 2, and the crystal form was determined and corrected by the method described in Example 3. As shown in Figures 5 through 1, the attachment of the inhibitor results in the removal of six water molecules in the active site pocket, including the water molecules attached to the zinc being replaced by the inhibitor's ρ 嗤 嗤 nitrogen. Inhibitors differ in their orientation due to the different changes in the 20 1362417 imidazole ring. The small ethylene fraction of μ vinyl imidazole does not interact with the active site of human QC, so it combines to catalyze the next large space ( Figure 5). However, the main hydrophobic benzene ring on which Ν-benzyl π is seated is closely surrounded and stabilized by the phenyl and hydrazine groups of F325 and W329, respectively (Fig. 5C). Conversely, the substituent orientation of Ν-ω-acetyl histamine is almost parallel to the backbone of fragment G301-304, and is mainly stabilized by three additional hydrogen bonds to D248 〇δ2, q3〇4 N and Q304 0 of the enzyme ( Figure 5D). The detailed three-dimensional structures of the human QC/1_: alkenyl imidazole complex, the human QC/N-nodal imidazole complex, and the human qc/n-^ acetyl histamine complex are stored in the protein database (website location www. Pdb.org), the protein database identifier (pDB is still c〇d (4) is characterized by the atomic structure coordinates of 2AFZ, 2AFX and 2AFW. Those who are familiar with this technology can understand that the concept can be found without departing from the broad concept of invention. The present invention is not limited to the specific embodiment of the invention, and it is intended to cover the modifications and the scope of the invention as set forth in the appended claims. A brief description of the formulas of the present invention will be described in conjunction with the description of the present invention. For the purpose of illustrating the present invention, the presently preferred specific embodiments are illustrated in the drawings. The invention is not limited to the arrangement and composition of the substrates. In the drawings: Figure 1 shows the x-ray coordinates of the human QC crystal structure at pH 6.5. Figure 2 shows the x-ray coordinates of the human QC crystal structure at pH 8 。. Figure 3A shows the human QC structure Figure 3B is a schematic view showing the human qC topology. Figure 3C is a perspective view showing the human qC catalytic region. Figure 4 shows the date of human acinamine cyclase and face amine decyl tributyl complex X-ray coordinates of the bulk structure. Figure 5 shows the structure of the active site of human QC in free form. Figure 5B shows the structure of the active site of human QC combined with the 1-ethylene-based saliva. 5C, 7F at the resolution (5) into the active site structure of human QC bound to NH m saliva. • s shows the active site structure of human QC bound to Ν-ω-acetyl histamine at a resolution of 1.64A. 1362417 Sequence Listing <110> Academia Sinica <120> Crystal Structure of Water Soluble Chromate Amine Cyclizing Enzyme <130> 682781-004TW <160>1 <170> Patentln version 3.3 <210> 1 <211> 361

<212> PRT <213> Homo sapiens <400> 1

Met Ala Gly Gly Arg His Arg Arg Val Val Gly Thr Leu His Leu Leu 1 5 l〇 15

Leu Leu Val Ala Ala Leu Pro Trp Ala Ser Arg Gly Val Ser Pro Ser 20 25 30

Ala Ser Ala Tip Pro Glu Glu Lys Asn Tyr His Gin Pro Ala lie Leu 35 40 45

Asn Ser Ser Ala Leu Arg Gin lie Ala Glu Gly Thr Ser lie Ser Glu 50 55 60

Met Trp Gin Asn Asp Leu Gin Pro Leu Leu lie Glu Arg Tyr Pro Gly 65 70 75 80

Ser Pro Gly Ser Tyr Ala Ala Arg Gin His lie Met Gin Arg lie Gin 85 90 95

Arg Leu Gin Ala Asp Trp Val Leu Glu lie Asp Thr Phe Leu Ser Gin 100 105 110

Thr Pro Tyr Gly Tyr Arg Ser Phe Ser Asn lie lie Ser Thr Leu Asn 115 120 125

Pro Thr Ala Lys Arg His Leu Val Leu Ala Cys His Tyr Asp Ser Lys 1362417 130 135 140

Tyr Phe Ser His Trp Asn Asn Arg Val Phe Val Gly Ala Thr Asp Ser 145 150 155 160

Ala Val Pro Cys Ala Met Met Leu Glu Leu Ala Arg Ala Leu Asp Lys 165 170 175

Lys Leu Leu Ser Leu Lys Thr Val Ser Asp Ser Lys Pro Asp Leu Ser 180 185 190

Leu Gin Leu lie Phe Phe Asp Gly Glu Glu Ala Phe Leu His Trp Ser 195 200 205

Pro Gin Asp Ser Leu Tyr Gly Ser Arg His Leu Ala Ala Lys Met Ala 210 215 220

Ser Thr Pro His Pro Pro Gly Ala Arg Gly Thr Ser Gin Leu His Gly 225 230 235 240

Met Asp Leu Leu Val Leu Leu Asp Leu lie Gly Ala Pro Asn Pro Thr 245 250 255

Phe Pro Asn Phe Phe Pro Asn Ser Ala Arg Trp Phe Glu Arg Leu Gin 260 265 270

Ala lie Glu His Glu Leu His Glu Leu Gly Leu Leu Lys Asp His Ser 275 280 285

Leu Glu Gly Arg Tyr Phe Gin Asn Tyr Ser Tyr Gly Gly Val lie Gin 290 295 300

Asp Asp His lie Pro Phe Leu Arg Arg Gly Val Pro Val Leu His Leu 305 310 315 320 lie Pro Ser Pro Phe Pro Glu Val Trp His Thr Met Asp Asp Asn Glu 325 330 335

Glu Asn Leu Asp Glu Ser Thr lie Asp Asn Leu Asn Lys lie Leu Gin 340 345 350

Val Phe Val Leu Glu Tyr Leu His Leu 355 360 2

Claims (1)

1362417 VII, the scope of application for patents: L A human acid amine cyclase (caffe, QC) crystal, its three-dimensional structure named qC8.0, with a space group of R32 to form a size of (four) = 118 99 into , the unit cell lattice. Such as the scope of the patent claim! The Qc crystal of the item wherein the crystal X-ray is diffracted to determine the atomic coordinate of the crystal at a resolution of about 2.35. 3. The qc crystal of claim 5, wherein the crystal comprises two QC molecules. 4. For the QC crystal described in claim 3, wherein all of the C atoms between the two QC molecules have a root mean square error of about 0.368 Α. 5. A human QC/(crystal of the JC matrix complex, comprising: (4) polymorphism, having a monthly female base sequence of 33 to 361 residues in SEQ ID NO: 1, or a homolog thereof or the like Or a variant, the Helicobacter peptide itself may form a QC crystal as claimed in any one of claims 4 to 4; and, (b) a QC matrix; wherein the complex & θ Μ > The effective X-ray diffraction of the body determines the atomic coordinates of the QC/QC matrix composite at a resolution of about 2.22 A. 6. The crystal of the composite according to claim 5, which has a space group of R32 to form a size. The unit cell of a=b=u9 14 A, c-332.61 A. 7. A method for identifying an inhibitor of human QC, comprising: (a) preparing a polypeptide having sequence identification number 1 in points 33 to 361 An amino acid sequence of an amino acid residue, wherein the polypeptide has an active site of a qC crystal according to any one of claims 1 to 4, wherein the active site comprises a zinc ion tetrahedron and a sequence identifier 159, 202 and 330 amino acid residues, and one water molecule coordination; (b) contacting the polypeptide to be tested An inhibitor to form a mammalian QC-inhibitor complex; and (c) a three-dimensional model for producing a mammalian qC_inhibitor complex obtained by the step; wherein the inhibitor to be tested having imidazole nitrogen and zinc ions is identified The method of claim 7, wherein the active site further comprises 201, 207, 248, 305, 325 and 329 amine groups in SEQ ID NO: 1. 9. The method of claim 7, wherein the active site further comprises a 159th amino acid residue of the 160th amino acid 1362417 of SEQ ID NO: 1 as a peptide bond Binding to the sequence identifier residue. 1〇. The method described in item 9 of the _li range, wherein the cough peptide bond is stabilized by a plurality of hydrogen bonds—the cis-form shape wins the bond. " U The method of claim 10, wherein the active site further comprises a storage buffer, the first identification number 144, 146, 154, 249 303 i, Chuan 3 , 321 , 325 and 329 amino acid residues 12. The method of claim n, wherein the active site further comprises a sulfate ion adjacent to the titanium water bag. The ϋ ϋ salt ion is identified by a hydrogen bonding sequence identification number 144, 2 〇 6, 207 and 33 胺 amino acid residues, and at least two water molecules 0. 13. A method of producing a crystal according to any one of claims 1 to 4, comprising: 0) expressing the (b) purifying the QC protein; and (4) crystallizing the (8) protein to form a crystal according to any one of claims 1 to 4. 14. The method of claim 5, wherein the QC protein is precipitated by a suspension diffusion method