Inhibition and inactivation of vanadium bromoperoxidase by the substrate hydrogen peroxide and further mechanistic studies

Abstract

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 (KiiH2O2, KisH2O2) versus H+ concentration indicates that an ionizable group with a pKa 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 H2(18)O2 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 O2 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.

Thymol Bromination – A Comparison between Enzymatic and Chemical Catalysis

“…[6] As far as the enzymatic process is concerned, the kinetic plot (Figure 2) shows that the reaction does not proceed any further after 3 h. The low substrate conversion (27 %) might be due to inactivation of the enzyme by hydrogen peroxide. To substantiate this hypothesis, the stability of the enzyme in the presence of high concentrations of H 2 O 2 was investigated, and indeed after 5 h of incubation, the enzyme lost 85 % of its initial activity, in agreement with the work by Soedjak and Butler, [33] who showed inactivation and inhibition at high peroxide concentrations (Figure 3). Table 2.…”

Controlled layer-by-layer immobilization of horseradish peroxidase

“…There is a consensus that, at higher concentrations, hydrogen peroxide acts as a substrate inhibitor by forming a dead end product (compound III in reaction 5). This substrate inhibition has also been observed with other peroxidases (Soedjak et al, 1995).…”

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

“…Photolysis of ROCl to • Cl and • RO has been observed (47,48). However, in vanadium-dependent haloperoxides, a radical-generating reaction has been ruled out in favor of a mechanism generating cationic bromine (30).…”