Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide

Abstract

Hypochlorous acid is the most powerful oxidant generated by neutrophils and is likely to contribute to the damage mediated by these inflammatory cells. The haem enzyme myeloperoxidase catalyses its production from hydrogen peroxide and chloride. 4-Aminobenzoic acid hydrazide (ABAH) is a potent inhibitor of hypochlorous acid production. In this investigation we show that, in the presence of hydrogen peroxide, ABAH irreversibly inactivates myeloperoxidase. ABAH was oxidized by myeloperoxidase, and kinetic analysis of the inactivation conformed to that for a mechanism-based inhibitor. Inactivation was exacerbated by concentrations of hydrogen peroxide greater than 50 ƁM and by the absence of oxygen. Hydrogen peroxide alone caused minimal inactivation. Reduced glutathione inhibited the oxidation of ABAH as well as the irreversible inhibition of myeloperoxidase. In the presence of oxygen, ABAH and hydrogen peroxide initially converted myeloperoxidase into compound III, which susequently lost haem absorbance. In the absence of oxygen, the enzyme was converted into ferrous myeloperoxidase and its haem groups were rapidly destroyed. We propose that myeloperoxidase oxidizes ABAH to a radical that reduces the enzyme to its ferrous intermediate. Ferrous myeloperoxidase reacts either with oxygen to allow enzyme turnover, or with hydrogen peroxide to give irreversible inactivation.