THE ROLE OF ARGININE-143 IN THE ELECTROSTATICS AND MECHANISM OF CU,ZNSUPEROXIDE-DISMUTASE - COMPUTATIONAL AND EXPERIMENTAL EVALUATION BY MUTATIONAL ANALYSIS

Cu,Zn superoxide dismutase protects cells from oxidative damage by rem oving superoxide radicals in one of the fastest enzyme reactions known . The redox reaction at the active-site Cu ion is rate-limited by diff usion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu,Zn superoxide dismutase, single-site mutants at t his position were investigated experimentally and computationally. Rat e constants for several Arg-143 mutants were determined at different p H and ionic strength conditions using pulse radiolytic methods and com pared to results from Brownian dynamics simulations. At physioloscal p H, substitution of Arg-143 by Lys caused a a-fold drop in rate, neutra l substitutions (Ile, Ala) reduced the rate about 10-fold, while charg e-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Posit ion 143 mutants showed pH dependencies not seen in other mutants. At l ow pH, the acidic residue mutations exhibited protonation/deprotonatio n effects. At high pH, all enzymes showed typical decreases in rate ex cept the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (>10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzy me was unaffected. Reaction simulations with Brownian dynamics incorpo rating electrostatic effects tested computational predictability of io nic strength dependencies of the wild-type enzyme and the Lys, Ile, an d Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the el ectrostatic attraction of the substrate is accurately modeled. Differe nces between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-14 3 is the single most important residue in Cu,Zn superoxide dismutase b oth electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143. (C) 1994 W iley-Liss, Inc.