Mechanism-based inactivation of gastric peroxidase by mercaptomethylimidazole.
The mechanism of inhibition of gastric peroxidase (GPO) activity by mercaptomethylimidazole (MMI), an inducer of gastric acid secretion, has been investigated. Incubation of purified GPO with MMI in the presence of H2O2 results in irreversible inactivation of the enzyme. No significant inactivation occurs in the absence of H2O2 or MMI, suggesting the involvement of peroxidase-catalysed oxidized MMI (MMIOX.) in the inactivation process. The inactivation follows pseudo-first-order kinetics consistent with a mechanism-based (suicide) mode. The pseudo-first-order kinetic constants at pH 8 are ki = 111 microM, 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 x min-1. Propylthiouracil also inactivates GPO activity in the same manner but its efficiency (k(inact./ki = 0.46 mM-1 x min-1) is about 10 times lower than that of MMI (k(inact./ki = 5 mM-1 x min-1). The rate of inactivation with MMI shows pH-dependence with an inflection point at 7.3, indicating the involvement in the inactivation process of an ionizable group on the enzyme with a pKa of 7.3. The enzyme is remarkably protected against inactivation by micromolar concentrations of electron donors such as iodide and bromide but not by chloride. Although GPO oxidizes MMI slowly, iodide stimulates it through enzymic generation of I+ which is reduced back to I- by MMI. Although MMIOX. 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 MMI inactivates catalytically active GPO by acting as a suicidal substrate.
Bandyopadhyay
U
U
Department of Physiology, Indian Institute of Chemical Biology, Calcutta.
Bhattacharyya
D K
DK
Banerjee
R K
RK
D000818:Animals; D005753:Gastric Mucosa / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008713:Methimazole / Q000494:pharmacology*; D008962:Models, Theoretical; D009195:Peroxidase / Q000037:antagonists & inhibitors*; D011441:Propylthiouracil / Q000494:pharmacology; D051381:Rats
D008713:Methimazole; D011441:Propylthiouracil; D006861:Hydrogen Peroxide; D009195:Peroxidase