Reactivity study on microperoxidase-8.
The catalytic activity of the microperoxidase-8/H(2)O(2) system toward tyramine and 3-(4-hydroxyphenyl)propionic acid has been determined in acetate buffer, pH 5.0. Operating with a strong excess of hydrogen peroxide, the rate-determining step of the reaction was substrate oxidation. Owing to the fast microperoxidase-8 degradation, only the very initial phase of the reactions were analyzed. The reaction rates follow a substrate saturation behavior, with turnover numbers [ k(cat)=26+/-1 s(-1) for 3-(4-hydroxyphenyl)propionic acid and k(cat)=22+/-1 s(-1) for tyramine] that were similar for the two substrates. In contrast, the K(M) values indicated a reduced affinity for the catalyst active species by the positively charged phenol, probably due to repulsive interaction with the protonated N-terminal microperoxidase-8 amino group. The reactivity of the catalyst active species was studied upon incubation of microperoxidase-8 with a small excess hydrogen peroxide, followed by reaction with the phenolic substrates. The kinetic analysis showed that more than two active species are accumulated. The species responsible for the faster reactions was present in solution as a minor fraction. The active intermediate which accumulated in a larger amount (intermediate III) has a reduced substrate oxidation activity. Comparison of this activity with the kinetic constants obtained under turnover experiments shows that intermediate III is not involved in the microperoxidase-8 catalytic cycle. The active species of the catalytic process are intermediates I and II, which in the absence of substrate rapidly convert to intermediate III.
D000818:Animals; D002384:Catalysis; D006736:Horses; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D010636:Phenols / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis; D014439:Tyramine / Q000737:chemistry / Q000378:metabolism
D010636:Phenols; D006861:Hydrogen Peroxide; D010544:Peroxidases; C021799:microperoxidase; D014439:Tyramine