MODULATION OF THE CATALYTIC RATE OF CU,ZN SUPEROXIDE-DISMUTASE IN SINGLE AND DOUBLE MUTANTS OF CONSERVED POSITIVELY AND NEGATIVELY CHARGED RESIDUES

The catalytic rate of four single and three double mutants of Xenopus laevis Cu,Zn superoxide dismutase B, neutralized at Lys120, Asp130, Gl u131, and Lys134, has been determined by pulse radiolysis as a functio n of ionic strength. Neutralization of Glu131 increases the catalytic rate by 80% at low ionic strength, but the effect is reduced to 50% at physiological ionic strength. The rate is unperturbed upon neutraliza tion of Asp130, while neutralization of either of the two lysines dras tically decreases the enzyme activity. The Lys 120Leu-Lys134Thr and Ly s134Thr-Asp130Gln double mutations have an additive and a compensative effect, respectively, on the activity values, while neutralization of the Glu131-Lys134 pair, which also has a compensative effect, gives r ise to a faster enzyme at any ionic strength value. The effects observ ed in the single Asp130Gln and Lys120Leu mutants differ from those rep orted on human or bovine enzymes [Getzoff et al. (1992) Nature (London ) 358, 347-351; Sines et al. (1990) Biochemistry 29, 9403-9412], indic ating that some residues occupying the same position in the linear seq uence of different Cu,Zn superoxide dismutases have a different functi onal weight. Our results also suggest that the strategy of multiple ch arge mutation may be a promising approach in order to increase the cat alytic rate of Cu,Zn SODs independently of ionic strength.