TUNING THE ACTIVITY OF CU,ZN SUPEROXIDE-DISMUTASE THROUGH SITE-DIRECTED MUTAGENESIS - A RELATIVELY ACTIVE MONOMERIC SPECIES

An enzymatically active monomeric analog of human copper,zinc superoxi de dismutase (SOD) was produced by replacing four hydrophobic residues at the dimer interface of wild-type SOD (WT) with hydrophilic residue s in a manner which has maintained the overall protein charge (i.e., P he50Glu, Glp51Glu, Val148Lys, Ile151Lys). This analog has been charact erized by (1) molecular weight determination, (2) several spectroscopi c techniques probing catalytic site geometry and (3) enzymatic activit y measurements at various ionic strengths. At physiological ionic stre ngth the present monomer has sizable activity being five times that of a previously reported monomeric analog carrying only two of these sub stitutions with an overall charge two units more negative than WT (i.e ., Phe50Glu, Gly51Glu). Unlike the catalytic activity of the latter an alog, this one reveals an ionic strength dependency like that of WT. E nzymatic behavior is discussed with regard to factors affecting substr ate diffusion towards the catalytic site.