Mechanism of horseradish peroxidase inactivation by benzhydrazide: A critical evaluation of arylhydrazides as peroxidase inhibitors

Abstract

Many compounds are oxidized by haem enzymes, such as peroxidases and cytochromes P450, to highly reactive intermediates that function as enzyme inactivators. To evaluate the potential of arylhydrazides as selective metabolically activated peroxidase inhibitors, the mechanism of HRPC (horseradish peroxidase isoenzyme C) inhibition by BZH (benzhydrazide) was investigated in detail. No oxygen consumption was detected in BZH solutions at pH 7.0-12.0, but addition of HRPC resulted in significant O2 uptake above pH 8.0, indicating that the enzyme catalyses BZH oxidation. Addition of H2O2 to HRPC plus BZH activates the latter as an inhibitor. This involves the three-electron oxidation of BZH in one-electron steps by the peroxidase catalytic intermediates, Compounds I and II, to produce a benzoyl radical that covalently alters the active site and inhibits peroxidase activity. Alternatively, the benzoyl radical could be produced by di-imide (NH = NH) elimination from the BZH radical. Production of Compound III (oxyperoxidase) followed by p-670 (m/z = 583, biliverdin-like derivative) was observed for HRPC incubated with excess H2O2, and the addition of BZH resulted in an increase in the rate of p-670 production. BZH is an inefficient inhibitor of HRPC with a K1 of 80 μM, an apparent inactivation rate constant (kinact) of 0.035 min-1, and an IC50 of 1.0 mM. This prompted the investigation of HRPC inactivation by a series of related arylhydrazides with known binding affinities for HRPC. The hydrazide with the highest affinity (2-naphthoichydrazide; Kd = 5.2 μM) was also found to be the most effective inhibitor with K1, Kinact and IC50 values of 14 μM, 0.14 min-1 and 35 μM, respectively.