[Inactivation of glutathione reductase by quinones and nitrosocarbamides]. / Inaktivatsiia gliutationreduktazy khinonami i nitrozokarbamidami.

Quinones inactivate oxidized glutathione reductase (EC 1.6.4.2) from yeast with rate constants (ki) ranging from 0.03 M-1s-1 (p-benzoquinone) to 10(3) M-1s-1 (bromanyl). It is glutathione, but not NADP+ that protects the enzyme from inactivation, which shows that quinones interact with a glutathione-binding centre, cysteine-2, most probably. The mechanism of inactivation by quinones differs from that by nitrosoureas which inactivate only the reduced enzyme, modifying the reduced catalytic disulphide. 1,3-bis-(2-chloroethyl)-1-nitrosourea acts as the most rapid inactivator of the enzyme, possessing ki of 0.77 M-1s-1.

[Nonphysiological redox-agents are reduced at the binding center of NADP(H) glutathione reductase]. / Nefiziologicheskie redoks-agenty vosstanavlivaiutsiia v tsentre sviazyvaniia NADP(H) glutationreduktazy.

Studies of the acceptor reductase reaction of yeast glutathione reductase (EC 1.6.4.2) revealed that the competitive inhibitors for NADPH, 2',5'-ADP and Br- decrease the rate constants for the enzyme oxidation by ferricyanide, phenanthrene quinone, and juglone. A similar effect is observed when NADH which does not bind to the reduced enzyme is used as substrate. These observations support the hypothesis that non-physiological redox agents are reduced at the NADP(H)-binding center of glutathione reductase and that NADP(H) binding stimulates the reaction by displacing tyrosine-197 which protects FAD from the solvent.

[Stimulation of the NADPH:adrenodoxin reductase diaphorase reaction by adrenodoxin]. / Stimulirovanie diaforaznoi reaktsii NADPH:adrenodoksinreduktazy adrenodoksinom.

The diaphorase activity of NADPH: adrenodoxin reductase (EC 1.18.1.2) is stimulated by adrenodoxin. The latter prevents the reductase inhibition by NADPH; the Line-weaver-Burk plots are characterized by a biphasic dependence of the reaction rate on the oxidizer concentration. At pH 7.0 the maximal rate of the first phase is 20s-1; that for the second phase at saturating concentrations of adrenodoxin is 5 s-1. Since the second phase rate is equal to that of the adrenodoxin-linked cytochrome c reduction by reductase it is concluded that this phase reflects the reduction of the oxidizers via reduced adrenodoxin. Quinones are reduced by adrenodoxin in an one-electron way; the logarithms of their rate constants depend hyperbolically on their single-electron reduction potentials (E7(1]. The oxidizers interact with a negatively charged domain of adrenodoxin. The depth of the adrenodoxin active center calculated from the Fe(EDTA)- reduction data is 5.9 A.

[Reaction of complex I of the mitochondrial electron transport chain with artificial oxidizers]. / Reaktsii kompleksa I mitokhondrial'noi tsepi transporta élektronov s iskusstvennymi okisliteliami.

The steady-state kinetics of oxidation of the mitochondrial NADH: ubiquinone oxidoreductase (complex I, EC 1.6.99.3) by artificial electron acceptors--p-quinones and inorganic complexes has been investigated. A limiting stage in the NADH: ferricyanide reductase reaction is a reductive half-reaction. Ferricyanide interacts with negative-charged protein groups taking part in the NADH binding. The rate constants of the quinone reduction by complex I vary from 1.10(6) to 4.10(3) M-1s-1. The NADH, NAD+ and ADP-ribose inhibition data indicate that oxidizers in the rotenono-insensitive reaction interact with the redox centre near the NAD+/NADH binding site, most probably with FMN.

[The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae]. / Sootnosheniia mezhdu glutationreduktaznoi i diaforaznoi aktivnost'iu glutationreductazy iz Saccharomyces cerevisiae.

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.

[Inhibition of adrenodoxin reductase by NADP+ and NADPH]. / Ingibirovanie adrenodoksinreduktazy NADP+ i NADPH.

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.

[Kinetics of adrenodoxin reductase oxidation by non-physiologic electron acceptors]. / Kinetika okisleniia adrenodoksinreduktazy nefiziologicheskimi aktseptorami élektronov.

The reactions of NADPH oxidation by quinones and inorganic complexes catalyzed by NADPH: adrenodoxin reductase were studied. The catalytic constant for the enzyme at pH 7.0 is 20-25 s-1; the oxidative constants for the quinones vary from 5 X 10(5) to 1.1 X 10(3) M-1 s-1 and show an increase with a rise in the one-electron acceptor reduction potential. The mode of adrenodoxin reductase interaction with oxyquinones differs from that of the enzyme interaction with alkyl-substituted quinones and inorganic complexes. NADPH competitively inhibits electron acceptors, whereas NADP+ is a competitive inhibitor of NADPH and a uncompetitive inhibitor of electron acceptors. (Ki = 25 microM). The depth of FAD incorporation into the enzyme molecule as calculated according to the outer sphere electron transfer theory is 6.1 A.

[Characteristics of the interaction of adrenal lipoamide dehydrogenase with physiological and quinone electron acceptors]. / Zakonomernosti vzaimodeistviia lipoamiddegidrogenazy nadpochechnikov s fiziologicheskimi i khinonovymi aktseptorami élektronov.

Lipoamide dehydrogenase (EC 1.6.4.3) from the ketoglutarate dehydrogenase complex of adrenals catalyzes the oxidation of NADH by lipoamide and quinone compounds according to the "ping-pong" scheme. The catalytic constants of these reactions are equal to 220 and 24 s-1, respectively (pH 7.0). The maximal quinone reductase activity is observed at pH 5.6, whereas the lipoamide reductase activity changes insignificantly at pH 7.5-5.5. The maximal dihydrolipoamide-NAD+ reductase activity is observed at pH 7.8. The oxidative constants of quinone electron acceptors vary from 6 X 10(6) to 4 X 10(2) M-1 s-1 and increase with their redox potential. The patterns of NAD+ inhibition in the quinone reductase reaction differ from that of lipoamide reductase reaction. The quinones are reduced by lipoamide dehydrogenase in the one-electron mechanism.

[Diaphorase reactions of lipoamide dehydrogenases from the adrenal ketoglutarate dehydrogenase complex]. / Diaforaznye reaktsii lipoamiddegidrogenazy iz oksoglutaratdegidrogenaznogo kompleksa nadpochechnikov.

Lipoamide dehydrogenase, a component of the bovine adrenal ketoglutarate dehydrogenase complex, catalyzes the oxidation of NADH by p-quinones and ferricyanide. The kinetics of oxidation obey the ping-pong mechanism. At pH 7.0, the constants for the active center oxidation by quinones (kox) are equal to 1.1 X 10(4)-5.3 X 10(5) M-1s-1 and increase as the acceptor potential rises. The values of kox for quinones change insignificantly within the pH range of 7.7-5.0, whereas that for ferricyanide increases 10-fold with a decrease of pH from 7.0 to 5.0. The value of the catalytic constant for the enzyme (kcat) reaches its maximum at pH 5.5. The quinones interact with the thiol groups of lipoamide dehydrogenase by inhibiting the fluorescence of FAD and diaphorease activity. The reaction is catalyzed by a basic amino acid (pK 6.7) within the composition of the enzyme.

[Enzymatic oxidation of glucose on modified electrodes]. / Fermentativnoe okislenie gliukozy na modifitsirovannykh élektrodakh.

Glucose oxidase (EC 1.1.3.4) entrapped at the paraelectrode layer of a glass-carbon electrode coated with tetracyano-p-quinodimethane, tetracyano-p-quinodimethane anion potassium salt, piocyanine. 9.10-phenantrenequinone, dichlorophenolindophenol or dextran-dopamine catalyzes electrochemical oxidation of glucose. The value of maximal current amounts to 67 micro A . cm-2 and depends on the enzyme concentration and the nature of the modifier. The compounds used are substrates for glucose oxidase. The electrocatalytical oxidation of glucose occurs in a mediator fashion.