Abstract

A protocol has been established for the over-expression and purification of large quantities of yeast enolase from E. coli cells. Yields have frequently exceeded 200 mg of >90% pure enzyme. Homogeneity of the preparations was assessed using both SDS-PAGE and analytical ultracentrifugation. Site-directed mutagenesis was successfully utilized to effect amino acid substitutions at two positions involved in subunit contacts. The residues in question are tryptophan 56, which was changed to phenylalamne (W56F), and glutamic acid 188, which was changed to aspartic acid (E188D). The mutants were characterized both structurally and catalytically, in order to study the effects of these substitutions on the enzyme. Both mutants are partially dissociated in the 'apo' form, and are significantly more readily dissociable than is wild type in the presence of NaClO 4 , a chaotropic salt. Each mutant shows a decreased conformational stability, or xG (H2O) , with respect to wild type. Binding of cofactor and substrate allow the mutants to associate more fully and protect against dissociation when incubated with NaClO 4 although the holoenzymes remain significantly more readily dissociable than wild type enolase. A survey of the mutants' kinetic properties reveals a decrease in k cat in the presence of the cofactors, Mg 2+ and Mn 2+ . The modified Mg 2+ inhibition reveals a change in metal ion specificity at enolase's third and inhibitory metal ion binding site. This finding in both mutants suggests that this site may lie in close proximity to the subunit interface, if not at the interface. The effects of these mutations on both structure and catalysis reveal the integral importance of dimerization for the enzyme's function, and, more specifically, the role of the Pro35-Gly60 loop in maintaining this delicate balance. (Abstract shortened by UMI.)