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Kinetic and e.p.r. studies on the inhibition of xanthine oxidase by alloxanthine (1 H-pyrazolo [3, 4-d] pyrimidine-4,6-diol).

ABSTRACT: The inhibition by alloxanthine of oxidation of xanthine by xanthine oxidase is characterized by a prolonged transient phase. Kinetic data accord with a mechanism that involves rapid formation of a reduced enzyme-alloxanthine complex that subsequently undergoes a relatively slow-reversible reaction. In this scheme the slowly formed complex cannot be fully reoxidized by oxygen. From the Ki value for the dissociation of alloxanthine from the rapidly formed complex (1.15 microM) and values of 0.37 min-1 and 0.011 min-1 for the forward and reverse rate constants of the slow reaction, an overall inhibition constant for alloxanthine of 35 nM was calculated. A molybdenum (V) e.p.r. signal from the slowly formed reduced enzyme-alloxanthine complex is described. The rate of appearance of this new signal is consistent with this assignment. The signal (the "Alloxanthine signal") was simulated with g1 2,0269, g2 1,9593, g3 11.9444 and shows indications of hyperfine coupling to nitrogen. Similarities between it and the Very Rapid signal are discussed. Close structural analogies between the catalytic intermediate represented by the Very Rapid signal and the inhibitor complex represented by the Alloxanthine signal are suggested.

SUBMITTER: Williams JW

PROVIDER: S-EPMC1162948 | biostudies-other | 1981 Jun

REPOSITORIES: biostudies-other

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The structure of the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,6-diol) with the molybdenum centre of xanthine oxidase from electron-paramagnetic-resonance spectroscopy.

Project description:Studies were carried out on the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,5-diol) with xanthine oxidase, in extension of the work of Williams & Bray [Biochem. J. (1981) 195, 753-760]. By suitable regulation of the reaction conditions, up to 10% of the functional enzyme could be converted into the complex in the Mo(V) oxidation state. The e.p.r. spectrum of the complex was investigated in detail with the help of computer simulation and substitution with stable isotopes. Close structural analogy of the signal-giving species to that of the Very Rapid intermediate in enzyme turnover is shown by g-values (2.0279, 1.9593 and 1.9442) and by coupling to 33S in the cyanide-labile site of the enzyme [A(33S) 0.30, 3.10 and 0.70mT]. However, whereas in the Very Rapid signal there is strong coupling to 17O [Gutteridge & Bray, Biochem. J. (1980) 189, 615-623], instead, in the Alloxanthine signal there is strong coupling to a single nitrogen atom [A(14N) 0.35, 0.35, 0.32 mT]. This is presumed to originate from the 2-position of the heterocyclic ring system. From this work and from earlier kinetic studies it is concluded that alloxanthine, after being bound reversibly at the active centre, reacts slowly with it, in a specific manner, distinct from that in the normal catalytic reaction with substrates. This reaction involves elimination of an oxygen ligand of molybdenum and co-ordination, in this site, of alloxanthine via the N-2 nitrogen atom, to give a complex that is structurally but not chemically closely analogous to that of the Very Rapid species.

| S-EPMC1153429 | biostudies-other

Inhibition studies of bovine xanthine oxidase by luteolin, silibinin, quercetin, and curcumin.

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Kinetic studies on protoporphyrinogen oxidase inhibition by diphenyl ether herbicides.

Project description:Diphenyl ethers (DPEs) and related herbicides are powerful inhibitors of protoporphyrinogen oxidase, an enzyme involved in the biosynthesis of haems and chlorophylls. The inhibition kinetics of protoporphyrinogen oxidase of various origins by four DPEs, (methyl)-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid (acifluorfen and its methyl ester, acifluorfen-methyl), methyl-5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-chlorobenzoate (LS 820340) and methyl-5-[2-chloro-5-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid (RH 5348), were studied. The inhibitions of the enzymes from maize (Zea mays) mitochondrial and etiochloroplastic membranes and mouse liver mitochondrial membranes were competitive with respect to the substrate, protoporphyrinogen IX, for all four molecules. The relative efficiencies of the inhibitors were: acifluorfen-methyl greater than LS 820340 much greater than RH 5348 greater than or equal to acifluorfen. The four molecules showed mixed-competitive type inhibition of the enzyme from yeast mitochondria where acifluorfen, a carboxylic acid, had the same inhibitory activity as its methyl ester, acifluorfen-methyl, and both were much greater than that of LS 820340 and RH 5348.

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2-Iodo-4,6-dimethyl-pyrimidine.

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Anticolon Cancer Properties of Pyrazole Derivatives Acting through Xanthine Oxidase Inhibition.

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