Inactivation of human arginine-143, lysine-143, and isoleucine-143 Cu,Zn superoxide dismutases by hydrogen peroxide: Multiple mechanisms for inactivation

Abstract

Site-specific mutants of human Cu,Zn superoxide dismutase (Cu,ZnSOD) have been prepared in which the active-site arginine at position 143 (i.e., SOD R143) has been replaced by either lysine (SOD R143) or isoleucine (SOD R143). As reported previously ( W. F. Beyer, Jr., et al. (1987) J. Biol. Chem. 262, 11182–11187), SOD K143 and SOD I143 have 43 and 11%, respectively, of the catalytic activity of SOD R143. H 2O 2, at low concentrations, acts as an affinity reagent for the inactivation of SOD R143. At pH 9.0 and 25 °C, the process is characterized by a half-saturation constant for H 2O 2, K 50, of 5.1 m m and a maximum pseudo-first-order rate constant for inactivation, k max, of 0.53 min −1. At pH 11.5, the corresponding values are 0.63 m m and 1.23 min −1. The active species in the inactivation is likely HO 2 −1, as previously found with yeast and bovine Cu,ZnSODs (see C. L. Borders, Jr., and I. Fridovich (1985) Arch. Biochem. Biophys. 241, 472–476). SOD K143 is also inactivated by HO 2 − by an affinity mechanism, i.e., one where reversible binding of H 2O 2 (HO 2 −) is a prerequisite for inactivation. At pH values of 9.0 and 11.5, the k max values are 0.92 and 1.08 min −1, respectively; however, the corresponding K 50 values increase to 42.5 and 15.8 m m, respectively. SOD I143 is also inactivated by H 2O 2, but no evidence for an affinity mechanism was found; instead, a second-order kinetic mechanism was observed. Inactivation of each of the three enzymes is accompanied by the loss of one histidine per subunit. At elevated concentrations of H 2O 2, a second nonaffinity mechanism of inactivation of both SOD R143 and SOD K143 was found, in which a second equivalent of H 2O 2 reacts with the Cu, ZnSOD · HO 2 − complex to give a competing second-order inactivation. It appears that the positive charge of arginine-143 plays a role in the binding of HO 2 − at the active site of human Cu,ZnSOD, and that replacement of the arginine by lysine gives an enzyme with a similar affinity mechanism of inactivation, but with a greatly reduced affinity for HO 2 −. However, replacement with isoleucine causes an entirely different mechanism of inactivation; this raises the possibility that the mechanism of enzyme catalysis of superoxide dismutation by SOD I143 is also different.