Transient-state and steady-state kinetics of the oxidation of aliphatic and aromatic thiols by horseradish peroxidase

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In the course of oxidation of thiols by peroxidases thiyl radicals are formed which are known to undergo several free-radical conjugative reactions, among others leading to hydrogen peroxide formation. The present paper for the first time presents a comparative transient-state and steady-state investigation of the reaction of 15 aliphatic and aromatic mono- and dithiols with horseradish peroxidase (HRP). Both sequential-stopped-flow spectrophotometric investigations of the reaction of HRP intermediates Compound I ( k 2) and Compound II ( k 3) with thiols and measurements of the overall thiol oxidation and the simultaneous oxygen consumption in the presence and absence of exogenously added hydrogen peroxide (10 μM) have been performed. With HRP as thiyl radical generator it was shown that three groups of thiols have to be distinguished: (i) Aromatic thiols (e.g. thiophenol, 2-mercaptopurine) were excellent electron donors of both Compounds ( k 2: 10 4–10 7 M −1 s −1 and k 3: 10 3–10 6 M −1 s −1); however, the overall reaction was shown to depend on addition of hydrogen peroxide, indicating insufficient peroxide regeneration by arylthiyl radicals. (ii) Aliphatic thiols which were extremely bad substrates ( k 3 <10 M −1 s −1) for HRP (e.g. homocysteine, glutathione) and/or have a p K a,SH >9.5 (e.g. N-acetylcysteine, α-lipoic acid) were also shown to depend on exogenously added H 2O 2 to maintain the peroxidasic reaction, whereas (iii) with those thiols with rates of k 3 between 11 and 1600 M −1 s −1 (e.g. cysteine, cysteamine, cysteine methyl ester, cysteine ethyl ester) and/or with a p K a,SH <8 (penicillamine) thiol oxidation was independent of exogenously added hydrogen peroxide, indicating sufficient hydrogen peroxide regeneration.