S.D. Varfolomeev

293 - Electrocatalysis of a cathodic oxygen reduction by laccase

Summary The effect of laccase adsorbed on carbon-black electrodes on the electrochemical reduction of molecular O2 has been studied. It is shown that laccase introduced into O2-saturated solution (acetate-phosphate buffer, pH=5,5) shifts the potential towards the positive side and accelerates the O2 electroreduction in the 1.2-0.6 V range. With the use of carbon-black adsorbed laccase, the potential reaches 1.2 V, which is close to the oxygen equilibrium potential. The dependence of the stationary potential on the pH and on the oxygen pressure indicates that the parameters ϖU/ϖpH and dU/d log po2 correspond to the coefficients in the Nernst equation for the O2−H2O couple. A scheme for the oxygen reduction with immobilized laccase isproposed on the basis of experimentally measured kinetic parameters.

Direct electron transfer effect biosensors

A review of biosensors based on the direct electron transfer effect is carried out in this work. Different redox enzymes and proteins using the direct electron transport mechanism for electrocatalytical processes are described. The tunneling of electrons as a possible explanation of the direct electron transfer effect is also discussed.

MECHANISM OF H2-ELECTROOXIDATION WITH IMMOBILIZED HYDROGENASE

Abstract The reaction of hydrogen electrooxidation on carbon-black electrodes with hydrogenase has been studied. It is shown that in the presence of hydrogenase a hydrogen equilibrium potential equal to 0.0 V is established on a carbon-black electrode in phosphate buffer, pH 7.5, saturated with hydrogen. The kinetic parameters have been studied and the mechanism of the hydrogen electroenzymic oxidation reaction has been determined.

192 - Electrochemical Properties of Peroxidase

The electrochemical behaviour of horse-radish peroxidase on gold-amalgamated and pyrographite electrodes has been studied using the potentiodynamic method. The enzyme exhibited reversible redox reactions in the potential range from −400 to −700 mV vs. the Ag|AgCl electrode. The electrochemical process observed has been studied as a function of the enzyme concentration and pH. The electrochemical reaction at pH < 4 was shown to result from redox reactions of hemin which is a product of peroxidase dissociation. The spectrophotometric analysis and the potentiodynamic behaviour of apoperoxidase showed that the electrochemical process within the range of stable hemin—apoperoxidase complex was not due to electron transfer to the active site of the enzyme. A mechanism of electrochemical reaction is proposed which includes reversible reduction—oxidation of the disulfide bonds.

Inhibition mechanism of Polyporus laccase by fluoride ion

This paper is devoted to a copper-containing oxidase-lactase @-diphenol: oxygen oxidoreductase, EC 1.10.3.2). It is one of the few enzymes that reduces O2 to Hz0 in a four-electron-transfer-process [ 11. We have studied the inhibition mechanism of lactase by Fand used the Fas an agent to clarify certain aspects of the catalytic mechanism of Polyponts laccase in general. Although the inhibition of lactase activity by Fwas discovered long ago [2] and this problem has been discussed [3-71, some aspects of inhibition are still unclear. It is well known that Fbinds with the type 2 Cu2+ of the lactase-active site [2]. We concentrated our attention on the following problems: (i) What is the nature of inhibition of lactase activity by F-? Is it a competitive or a non-competitive inhibitor with respect to an electron donor and 02? Answers to these questions could give us information about the role of type 2 Cu2+ in binding the electron donor or OZ. (ii) In what way does lactase activity and the effectiveness of lactase inhibition by Fdepend on pH?

Bioelectrocatalysis. Hydrogenase as catalyst of electrochemical hydrogen ionization

Abstract The bioelectrocatalysis (acceleration of electrochemical reactions in the presence of enzymes) is discussed. The central point of bioelectrocatalysis is given by an efficient electron transport mechanism from the active center of the enzyme to an electrode. In principle, this could be realized by direct electron exchange between the active center of the enzyme and the electrode and also by an intermediate electron transport to a labile mediator of low molecular weight. The mediator mechanism of electron transport between the active center of the enzyme and a macroelectrode is discussed in detail. The requirements to such mediators are summarized. As a model, the system hydrogen-hydrogenase-methylviologen-pyrographite electrode was investigated. In this system one may kinetically ideally organize the electrochemical hydrogen oxidation by transforming the chemical energy of the fuel into an electrochemical potential, which enables one to realize the anodic oxidation of hydrogen on carbon electrodes. The mechanism of bioelectrocatalysis involves enzymatic reduction of methylviologen with molecular hydrogen and electrochemical oxidation of the mediator. The kinetics of the enzymatic and of the electrochemical reaction steps were studied. On the pyrographite electrode the reversible electrochemical oxidation and reduction of the mediator were possible with the kinetics being limited by the diffusion of the reagent. In this case a reversible redox hydrogen potential was established on the electrode. The dependences of the limiting anodic currents of hydrogen oxidation on the enzyme and on the mediator concentrations are investigated. Kinetical models describing the effects observed are proposed.

Purification of prostaglandin H synthetase and a fluorometric assay for its activity

Prostaglandin H synthetase (PGH synthetase) has been purified to homogeneity from sheep vesicular glands. The pure enzyme has a specific activity of about 40 microM of arachidonic acid consumed per minute per milligram of protein, which corresponds to a turnover number of 2800 min-1 per subunit. The purified enzyme was obtained by one-stage chromatography on DEAE-Toyopearl 650 from Tween 20-solubilized microsomes. A sensitive fluorometric assay for PGH synthetase activity using homovanillic acid (HVA) as electron donor has been proposed. It has been shown that homovanillic acid may be used as the electron donor and that in the presence of HVA the enzyme has an activity of approximately 40 microM/min/mg.

Supercooperativity in platelet aggregation: Substituted pyridyl isoxazoles, a new class of supercooperative platelet aggregation inhibitors

The phenomenon of supercooperativity in platelet aggregation is manifested by the occurence of clear‐cut thresholds in dose—response relationships; in such cases the Hill coefficient has unusually high values. Approximation, by the Hill equation, of the relationship of the rate of arachidonate‐induced platelet aggregation to the concentrations of either the inducer or inhibitors such as substituted pyridyl isoxazoles (synthesized by us), indomethacin, and pinane thromboxane A2, demonstrated that the Hill coefficients ranged from 30 to 100. 3‐(3‐Pyridyl)‐5‐phenylisoxazole, which exhibited maximal anti‐aggregatory activity among the synthesized compounds, inhibited neither cyclooxygenase nor thromboxane synthase. The compounds affected the signal transduction pathway at/or posterior to the stage of thromboxane A2 reception.

Bioelectrocatalysis. Activation of a cathode oxygen reduction in the peroxidase-mediator carbon electrode system.

At the present time effects are known that increase the rates of electrode processes in electrochemical systems in the presence of biological catalysts. Thus the rate of glucose oxidation increases in the presence of immobilized glucose oxidase [ 1,2] . With bacterial hydrogenases electrochemical hydrogen ionization may occur on carbon electrodes at a rate close to that observed in the presence of the ‘classical’ catalyst, metallic platinum [3]. Recently a range of such acceleration effects of the electrode processes with biocatalysts was established. The effect of bioelectrocatalysis is to increase the rate of an electrochemical reaction by the presence of enzymes. Interest in bioelectrocatalytical effects was considerably stimulated by the possibility of creating electrochemical transformers, that may oxidize different fuels and generate an electrochemical potential [3]. The main problem in the bioelectrocatalysis is the conjugation of enzymatic and electrochemical reaction steps. In general this may take place via the following two mechanisms:

Hierarchical classification of hydrolases catalytic sites

UNLABELLED Universal ontology of catalytic sites is required to systematize enzyme catalytic sites, their evolution as well as relations between catalytic sites and protein families, organisms and chemical reactions. Here we present a classification of hydrolases catalytic sites based on hierarchical organization. The web-accessible database provides information on the catalytic sites, protein folds, EC numbers and source organisms of the enzymes and includes software allowing for analysis and visualization of the relations between them. AVAILABILITY http://www.enzyme.chem.msu.ru/hcs/