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 peroxide at the active-site Cu ion in a reaction that is rate-limited by diffusion and enhanced by electrostatic guidance. SOD has evolved to be one of the fastest enzymes known (V(max) approximately 2 x 10(9) M-1 s-1). The new crystal structures of human SOD show that amino-acid site chains that are implicated in electrostatic guidance (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.
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
E D
ED
Getzoff
D E
DE
Cabelli
C L
CL
Fisher
H E
HE
Parge
M S
MS
Viezzoli
L
L
Banci
R A
RA
Hallewell
D000595:Amino Acid Sequence; D003198:Computer Simulation; D004563:Electrochemistry; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D011487:Protein Conformation; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D005971:Glutamates; D018698:Glutamic Acid; D013482:Superoxide Dismutase; D008239:Lysine