A MODEL STUDY ON THE EFFECT OF AN AMINO GROUP ON THE ANTIOXIDANT ACTIVITY OF GLUTATHIONE-PEROXIDASE

In order to investigate mechanistic roles of the amino nitrogens locat ed at the active center of glutathione peroxidase (GPX), a selenium-co ntaining antioxidant enzyme, kinetic analyses and characterization of the intermediates of the model reaction (H2O2 + 2PhSH - 2H2O + PhSSPh) catalyzed by di-2-(N-cyclohexyl,N-(methylamino)methyl)phenyl diseleni de (1) have been performed. The rate equation in methanol at 25-degree s-C by changing the initial concentrations of H2O2 and catalyst 1 sugg ests that the model catalyst (1) behaves precisely like GPX in the red uction catalytic cycle. On the basis of the Se-77 NMR experiments in a 1:1 mixture of CD3OD and CDCl3 under rigorous nitrogen atmosphere, th ree intermediates, selenenyl sulfide 4, selenolate 5', and selenenic a cid 6, are characterized. Three specific roles of proximate nitrogen a toms on the GPX-like activity are proposed. (1) Both theory (MO calcul ation) and experiments (Se-77 NMR) suggest that the proximate nitrogen base activates the selenol intermediate (5) into the corresponding se lenolate anion (5', Se-77 NMR observed at delta 22 ppm), which should play a key role in accelerating the catalytic cycle. (2) The proximate nitrogen moiety stabilizes otherwise elusive selenenic acid intermedi ate (6), which is observable by Se-77 NMR (delta 1173 ppm). It is sugg ested by this direct observation of the selenenic acid intermediate th at intramolecular Se-N interaction prevents its deterioration by furth er oxidation in the catalytic system. (3) Since the formation of an Se -N hypervalent bonding is demonstrated by low-temperature dynamic H-1 NMR experiment for the selenenyl sulfide intermediate (4), it is expec ted that the nucleophilic attack of benzenethiol (PhSH) should occur p referentially at the sulfur atom of 4, allowing effective production o f the selenolate intermediate (5') in the catalytic cycle.