MECHANISM-BASED INACTIVATION OF GASTRIC PEROXIDASE BY MERCAPTOMETHYLIMIDAZOLE

The mechanism of inhibition of gastric peroxidase (GPO) activity by me rcaptomethylimidazole (MMI), an inducer of gastric acid secretion, has been investigated. Incubation of purified GPO with MMI in the presenc e of H2O2 results in irreversible inactivation of the enzyme. No signi ficant inactivation occurs in the absence of H2O2 or MMI, suggesting t he involvement of peroxidase-catalysed oxidized MMI (MMI(ox.)) in the inactivation process. The inactivation follows pseudo-first-order kine tics consistent with a mechanism-based (suicide) mode. The pseudo-firs t-order kinetic constants at pH 8 are k(i) = 111 muM, k(inact) = 0.55 min-1 and t1/2 = 1.25 min, and the second-order rate constant is 0.53 x 10(4) M-1.min-1. Propylthiouracil also inactivates GPO activity in t he same manner but its efficiency (k(inact)/k(i) = 0.46 mM-1.min-1) is about 10 times lower than that of MMI (k(inact)/k(i) = 5 mM-1.min-1). The rate of inactivation with MMI shows pH-dependence with an inflect ion point at 7.3, indicating the involvement in the inactivation proce ss of an ionizable group on the enzyme with a pK(a) of 7.3. The enzyme is remarkably protected against inactivation by micromolar concentrat ions of electron donors such as iodide and bromide but not by chloride . Although GPO oxidizes MMI slowly, iodide stimulates it through enzym ic generation of I+ which is reduced back to I- by MMI. Although MMI(o x.) is formed at a much higher rate in the presence of I-, a constant concentration of I maintained via the reduction of I+ by MMI, protects the active site of the enzyme against inactivation. We suggest that M MI inactivates catalytically active GPO by acting as a suicidal substr ate.