Oxidative inactivation of gastric peroxidase by site-specific generation of hydroxyl radical and its role in stress-induced gastric ulceration

Abstract

We have shown earlier that restraint-cold stress-induced gastric ulceration in rats is caused by metal ion dependent generation of hydroxyl radical (OH^∗) and oxidative inactivation of the gastric peroxidase (GPO), an important H₂O₂ scavenging enzyme. To study the mechanism of the oxidative damage of GPO, the purified enzyme was exposed to an OH^∗ generating system containing Cu²⁺, ascorbate, and H₂O₂. Kinetic studies indicate that the enzyme is inactivated in a time-dependent process showing saturation with respect to Cu²⁺ concentration. The enzyme specifically 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 superoxide dismutase, suggesting no role of superoxide. The rate of inactivation is not increased in D₂O excluding the involvement of singlet oxygen in the process. However, OH^∗ scavengers such as benzoate or mannitol cannot prevent inactivation. The results indicate a plausible generation of OH^∗ within the enzyme molecule as the cause of inactivation. Fragmentation of peptide linkage or intramolecular crosslinking, gross change of tertiary structure, or change in intrinsic tryptophan fluorescence which occurs in "global" oxidation are not evident. Inactivation is dependent on pH and from a plot of K_obs of inactivation against pH, the controlling role of an ionizable group of the enzyme having a pka of 7.8 could be suggested, deprotonation of which favors inactivation. Amino acid analysis shows a specific loss of two lysine residues in the inactivated enzyme. Competitive kinetic studies indicate that pyridoxal phosphate, a specific modifier of the lysine residue, prevents inactivation by competing with Cu²⁺ for binding at the GPO. A Cu²⁺ binding motif consisting at least of two lysine residues exists in GPO, which specifically binds Cu²⁺ and generates OH^∗. The radical oxidizes the lysine residues and perturbs the heme environment to cause inactivation. We suggest that oxidative damage of GPO is mediated by site-specific generation of OH^∗ and not by the OH^∗ generated in the bulk phase.