INHIBITION AND INACTIVATION OF VANADIUM BROMOPEROXIDASE BY THE SUBSTRATE HYDROGEN PEROXIDE AND FURTHER MECHANISTIC STUDIES

摘要

Hydrogen peroxide, which is a substrate of vanadium bromoperoxidase (V-BrPO), has been shown to be a noncompetitive inhibitor of V-BrPO. Hydrogen peroxide inhibition increases with increasing pH. The inhibition is reversible under the conditions of the initial steady-state kinetic experiments. Analysis of the inhibition constants (K-ii(H2O2),K-is(H2O2)) versus H+ concentration indicates that an ionizable group with a pK(a) between 6.5 and 7 is involved in the inhibition. The origin of the oxygen atoms in the dioxygen produced by the V-BrPO-catalyzed bromide-assisted disproportionation of hydrogen peroxide has been shown through (H2O2)-O-18 labeling experiments to originate from the same molecule of hydrogen peroxide. V-BrPO-catalyzed bromination is shown to be an electrophilic (Br+) as opposed to a radical (Br-.) process. The stoichiometry of H2O2 consumed to MCD reacted or to O-2 produced is reported. The concentration of hydrogen peroxide also affects the competition of dioxygen formation during MCD bromination; competitive dioxygen formation is strongly enhanced at high pH. Turnover of V-BrPO under conditions of very high hydrogen peroxide concentration leads to irreversible inactivation at pH 4 and pH 5. Much less inactivation occurs during turnover at long reaction times at higher pH (>pH 6), and the inactivation can be fully reversed by subsequent addition of vanadate.