FASTER SUPEROXIDE-DISMUTASE MUTANTS DESIGNED BY ENHANCING ELECTROSTATIC GUIDANCE

THE enzyme Cu, Zn superoxide dismutase (SOD) protects against oxidative damage by dismuting the superoxide radical O2.- to molecular oxygen and hydrogen peroxide1-3 at the active-site Cu ion4,5 in a reaction that is rate-limited by diffusion3,6 and enhanced by electrostatic guidance7-10. SOD has evolved to be one of the fastest enzymes known (V(max) approximately 2 x 10(9) M-1 s-1)6,11. The new crystal structures of human SOD12 show that amino-acid site chains that are implicated in electrostatic guidance8 (Glu 132, Glu 133 and Lys 136) form a hydrogen-bonding network. Here we show that site-specific mutants that increase local positive charge while maintaining this orienting network (Glu --> Gln) have faster reaction rates and increased ionic-strength dependence, matching brownian dynamics simulations incorporating electrostatic terms. Increased positive charge alone is insufficient: one charge reversal (Glu --> Lys) mutant is slower than the equivalent charge neutralization (Glu --> Gln) mutant, showing that the newly introduced positive charge disrupts the orienting network. Thus, electrostatically facilitated diffusion rates can be increased by design, provided the detailed structural integrity of the active-site electrostatic network is maintained.