Reactivity study on microperoxidase-8

The catalytic activity of the microperoxidase-8/
H2O2 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 [kcat=
26±1 s)1 for 3-(4-hydroxyphenyl)propionic acid and
kcat=22±1 s)1 for tyramine] that were similar for the
two substrates. In contrast, the KM 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.