OXIDATIVE INACTIVATION OF GASTRIC PEROXIDASE BY SITE-SPECIFIC GENERATION OF HYDROXYL RADICAL AND ITS ROLE IN STRESS-INDUCED GASTRIC-ULCERATION

We have shown earlier that restraint-cold stress-induced gastric ulcer ation in rats is caused by metal ion-dependent generation of hydroxyl radical (OH.) and oxidative inactivation of the gastric peroxidase (GP O), an important H2O2 scavenging enzyme. To study the mechanism of the oxidative damage of GPO, the purified enzyme was exposed to an OH. ge nerating system containing Cu2+, ascorbate, and H2O2. Kinetic studies indicate that the enzyme is inactivated in a time-dependent process sh owing saturation with respect to Cu2+ concentration. The enzyme specif ically requires Cu2+ and is not inactivated by the same concentration of Fe2+, Mn2+, or Zn2+. Sensitivity to catalase indicates the critical role of H2O2 in the inactivation. Inactivation is insensitive to supe roxide dismutase, suggesting no role of superoxide. The rate of inacti vation is not increased in D2O excluding the involve ment of singlet o xygen in the process. However, OH. scavengers such as benzoate or mann itol cannot prevent inactivation. The results indicate a plausible gen eration of OH. within the enzyme molecule as the cause of inactivation . Fragmentation of peptide Linkage or intramolecular crosslinking, gro ss change of tertiary structure, or change in intrinsic tryptophan flu orescence which occurs in ''global'' oxidation are not evident. Inacti vation is dependent on pH and from a plot of K-obs of inactivation aga inst pH, the controlling role of an ionizable group of the enzyme havi ng a pka of 7.8 could be suggested, deprotonation of which favors inac tivation. Amino acid analysis shows a specific loss of two lysine resi dues in the inactivated enzyme. Competitive kinetic studies indicate t hat pyridoxal phosphate, a specific modifier of the lysine residue, pr events inactivation by competing with Cu2+ for binding at the GPO. A C u2+ binding motif consisting at least of two lysine residues exists in GPO, which specifically binds Cu2+ and generates OH.. The radical oxi dizes the lysine residues and perturbs the heme environment to cause i nactivation. We suggest that oxidative damage of GPO is mediated by si te-specific generation of OH. and not by the OH. generated in the bulk phase. (C) 1998 Elsevier Science Inc.