CN102321607A - Method for improving catalytic activity of adenosine phosphate hydrolase through alternating electric field - Google Patents
Method for improving catalytic activity of adenosine phosphate hydrolase through alternating electric field Download PDFInfo
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- CN102321607A CN102321607A CN201110264352A CN201110264352A CN102321607A CN 102321607 A CN102321607 A CN 102321607A CN 201110264352 A CN201110264352 A CN 201110264352A CN 201110264352 A CN201110264352 A CN 201110264352A CN 102321607 A CN102321607 A CN 102321607A
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Abstract
The invention discloses a method for further improving activity of enzymatic adenosine phosphate hydrolysis reaction through stimulating enzyme by using alternating electric field. The method comprises the following steps: carrying out hydrolysis reaction in an electrolytic tank, and applying the peak voltage of 5 V and the frequency of 1600 Hz on adonosine triphosphate and adenosine diphosphate hydrolysis systems in the presence of adonosine triphosphate/ adenosine diphosphate enzyme. The substrate, the product and enzyme molecules are subject to polarization and are forced to migrate under alternating electric field, so that the probability that the substrate molecules penetrate into enzyme active sites is increased; simultaneously, the product molecules are promoted to leave the enzyme active sites, so that the rate of enzyme hydrolysis reaction is further increased. The method has the advantages of simple operation and no pollution, and can be further applied in the fields of enzyme engineering, electromagnetic pollution research and medical detection.
Description
Technical field
The present invention relates to a kind of method for catalyzing chemical reaction.Specifically relate to adopt alternating electric field to improve the process method of the catalytic activity of phosphoric acid adenosinhydrolase.
Background technology
Enzyme is a kind of biological catalyst efficiently.Even so, further improving enzymatic activity with the artificial method and remain the target that enzyme engineering is pursued, also is simultaneously a kind of important means that modern zymetology is probed into enzyme catalysis mechanism.
Discover that combining site in the zymoprotein structure and catalytic site are not inflexible (Hammes-Schiffer S.Biochemistry 2002,41,13335-13343; Eisenmesser EZ, Science 2002,295,1520-1523).Except the flexibility of protein conformation can produce many conformation substates, it is because inducing of substrate molecule could be agreed with the substrate molecule perfection that reactive site has more than.In fact, do not having under the situation of substrate, proteinic conformation will be changed (Eisenmesser EZ, et al.Nature 2005,438,117-121 between different substates; Huang, Y.P., et al.Nature 2005,438,36-37), substrate passes in and out reactive site in this conversion.If in reaction system, add an alternating electric field, substrate, product and protein molecule will polarize along the direction of electric field, and move to electrode.If further apply alternating electric field, substrate and product molecule will carry out vibratory drive.When the frequency of vibrational frequency and turnover reactive site near the time just possibly promote the catalytic capability of enzyme.Because electric field can exert an influence to ion, so alternating electric field and various ionic channel and corresponding biomembranous interaction are conspicuous (Liu DS, et al.J.Biol.Chem.1990,265,7260-7267; Markin VS., et al.J.Membr.Biol.1992,126:137-145).But existing influence also is not confirmed electric field as yet to lytic enzyme.
On the other hand, light current magnetic field to the influence of organism the same with noise possibly cause the public's health seriously influence (Weaver JC, Astumian RD.Science 1990,247,459-462).Cell or organism have possessed the ability of the extremely weak mechanical vibration of impression, electromagnetism periodic signal and lower concentration chemistry messenger molecule after long-term evolution, and can be to being responded by the small sample perturbations that these signals produced.This mechanism can be with zymoprotein to the response of alternating electric field as the basis.
Summary of the invention
One of the object of the invention is to set up a kind of enzyme catalysis environment method of improving.
Two of the object of the invention is for the biological effect of studying electromagnetic pollution a kind of chemical experiment method to be provided.
Three of the object of the invention is for the catalyst mechanism of studying enzyme a kind of experimental technique to be provided.
In order to achieve the above object; The present invention has set up a kind of system that is applicable to adenosine triphosphate and Adenosine diphosphate glucoside hydrolysis reaction under phosphoric acid adenosinhydrolase (being adenosine triphosphate/di-phosphate adenosinase) catalysis, adopts alternating electric field to accelerate the process method of speed of reaction.
Concrete technology is following:
Adenosine triphosphate/di-phosphate adenosinase with deriving from yam is investigated aforesaid method.But the hydrolysis of this enzyme catalysis adenosine triphosphate sodium discharges Adenosine diphosphate glucoside and phosphoric acid, and further catalysis then discharges single phosphoric acid gland glucoside and phosphoric acid.But through the burst size to the measurement on-line monitoring phosphoric acid of pH value of reaction system, thereby the estimation reaction process over time.
Two platinum electrodes are placed reaction system, and electrode is connected on the function signal generator.The frequency of regulating alternating electric field is 10～10
6In the Hz scope, in strength of electric field 1～20V/cm scope, the waveform of alternating electric field is sinusoidal wave.System temperature maintains 37 ℃.Concentration of phosphoric acid is to measure system pH by acidometer to calculate that every interval 5min measures a pH value; Electricity irritation continues 40min, adds the enzyme inhibitors stopped reaction then, and measures final state pH value.
Enzymic hydrolysis adenosine triphosphate and Adenosine diphosphate glucoside generate single phosphoric acid gland glucoside and phosphoric acid.Under the enzyme catalysis condition of routine, the hydrolysis of adenosine triphosphate and Adenosine diphosphate glucoside is spontaneous carrying out.Electric field does not influence the chemical equilibrium of reaction.But it will change speed of reaction through the interaction with activated energy barrier.Rate determining step for substrate, activated energy barrier is in oscillatory regime.
Mensuration result shows, the tendency that the catalytic activity of phosphoric acid adenosinhydrolase occurs falling after rising along with the rising of the peak voltage that adds alternating electric field.In other words, strength of electric field begins from 1V/cm, and the reaction rate constant under the enzyme catalysis increases with the rising of strength of electric field.When strength of electric field reached 5V/cm, speed of reaction reached maximum value.Further increase strength of electric field, speed of reaction demonstrates downward trend on the contrary.This is because in a single day high field intensity surpasses the electropotential of redox reaction, just can cause the electrochemical reaction of product, thereby disturb test result.In addition, the catalytic activity of phosphoric acid adenosinhydrolase is along with the rising of alternating electric field frequency also demonstrates a peak.When frequency was 6500Hz, speed of reaction reached maximum value.In the hydrolytic process of phosphoric acid adenosinhydrolase catalysis adenosine triphosphate and Adenosine diphosphate glucoside; At strength of electric field 5V/cm; Under the sinusoidal alternating electric field action of frequency 6500Hz; Compare with no electrical field stimulation, its rate constant rises to 2.94 μ mol/mgmin from 1.82 μ mol/mgmin, and the activity of enzyme is stimulated to have raise 61.5%.And the reaction system of no enzyme is not observed the raising of speed of reaction under this alternating electric field stimulates.Therefore visible, electrocatalysis can further improve the catalytic activity of phosphoric acid adenosinhydrolase, and this effect has dependency to strength of electric field and alternative frequency.
The hydrolysis reaction of adenosine triphosphate and Adenosine diphosphate glucoside has very big free energy to drive, and Δ G is approximately-15kT, so reaction does not need energy supply.Alternating electric field does not influence thermodynamics of reactions, has just promoted the vibration that reaction activity is built on the kinetics.
The invention has the advantages that:
1. alternating electric field can further improve the catalytic activity of lytic enzyme.More easily to the reactive site diffusion of enzyme molecule, product also is easy to from the enzyme molecule, diffuse out through the electricity irritation substrate.Electric field also produces certain influence to the enzyme conformation simultaneously.
2. method of the present invention can be to the EM field Pollution Study, and the researchs such as effect of electric field pair cell and organism provide experiment basis.
3. method of the present invention can provide experiment basis to the research of ionic channel.
4. method of the present invention can provide experiment basis to the enzymic catalytic reaction Study on Mechanism.
Embodiment
Embodiment 1:
The adenosine triphosphate that derives from yam/di-phosphate adenosinase (grade VI, lyophilized powder,＞200unit/mg protein) is joined the MgCl that contains 0.1% nonionogenic tenside NP40 and 4mM
2Histidine buffering liquid in (30mM, the pH value is 7.4).Then, the substrate adenosine triphosphate sodium that adds 4mM causes hydrolysis reaction, utilizes the pH value of the every interval of acidometer 5min monitoring and recording system, in order to follow the tracks of the release of phosphoric acid.The stimulation of alternating electric field is carried out in being inserted with the 4mm cuvette of two platinum electrodes, and electrode is connected to function signal generator, and the frequency of alternating electric field is 6500Hz, and strength of electric field is 5V/cm, and waveform is sinusoidal wave.System temperature maintains 37 ℃.Enzymic catalytic reaction continues 40min, adds the trichoroacetic acid(TCA) stopped reaction then, measures final state.
Each data point is the MV of 3 determination datas.Corresponding reaction rate constant comes from the calculating of linear least square homing method to the curve initial slope.
Embodiment 2:
Adenosine triphosphate/di-phosphate adenosinase is joined the MgCl that contains 0.1% nonionogenic tenside NP40 and 4mM
2The 30mM histidine buffering liquid in (the pH value is 7.4).Then, the substrate adenosine triphosphate sodium glucoside that adds 4mM causes hydrolysis reaction, utilizes the pH value of the every interval of acidometer 5min monitoring and recording system, in order to follow the tracks of the release of phosphoric acid.System temperature maintains 37 ℃.Enzymic catalytic reaction continues 40min, adds the trichoroacetic acid(TCA) stopped reaction then, measures final state.
Each data point is the MV of 3 determination datas.Corresponding reaction rate constant comes from the calculating of linear least square homing method to the curve initial slope, is designated as the blank reaction of no electrical field stimulation.
Embodiment 3:
At the MgCl that contains 0.1% nonionogenic tenside NP40 and 4mM
2The 30mM histidine buffering liquid in (the pH value is 7.4), the substrate adenosine triphosphate sodium that adds 4mM causes hydrolysis reaction, utilizes the pH value of the every interval of acidometer 5min monitoring and recording system, in order to the release of tracking phosphoric acid.The stimulation of alternating electric field is carried out in being inserted with the 4mm cuvette of two platinum electrodes, and electrode is connected to function signal generator, and the frequency of alternating electric field is 6500Hz, and strength of electric field is 5V/cm, and waveform is sinusoidal wave.System temperature maintains 37 ℃.Enzymic catalytic reaction continues 40min, adds the trichoroacetic acid(TCA) stopped reaction then, measures final state.
Each data point is the MV of 3 determination datas.Corresponding reaction rate constant comes from the calculating of linear least square homing method to the curve initial slope, is designated as the blank reaction of no enzyme.
Claims (3)
1. process method that adopts alternating electric field to improve enzymatic hydrolysis reaction speed; It is characterized in that: described process method is in the hydrolysis reaction of reactor drum, to apply a kind of alternating electric field, impels the raising of hydrolysis reaction through stimulating substrate and the motion of product organic molecule in zymophore.
2. a kind of process method that adopts alternating electric field to improve enzymatic hydrolysis reaction speed according to claim 1, it is characterized in that: the peak voltage that adds of alternating electric field is 5V, and frequency is 1600Hz, waveform is sinusoidal wave.
3. a kind of process method that adopts alternating electric field to improve enzymatic hydrolysis reaction speed according to claim 1, it is characterized in that: the substrate organic molecule is adenosine triphosphate and Adenosine diphosphate glucoside, and biological catalyst is the phosphoric acid adenosinhydrolase.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108328935A (en) * | 2018-04-16 | 2018-07-27 | 中国工程物理研究院激光聚变研究中心 | Alternating electric field auxiliary optical component surface etching treatment device and processing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108328935A (en) * | 2018-04-16 | 2018-07-27 | 中国工程物理研究院激光聚变研究中心 | Alternating electric field auxiliary optical component surface etching treatment device and processing method |
| CN108328935B (en) * | 2018-04-16 | 2024-02-27 | 中国工程物理研究院激光聚变研究中心 | Alternating electric field auxiliary optical element surface etching treatment device and treatment method |
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Application publication date: 20120118 |