Changing the metal ion selectivity of rabbit muscle enolase by mutagenesis: effects of the G37A and G41A mutations.

During the reaction catalyzed by enolase, a mobile loop, residues 36-45, closes over the active site. In order to probe the role of this loop movement in catalysis, the glycines at positions 37 and 41 of rabbit muscle enolase (beta beta) have been mutated to alanines. The mutant forms-G37A and G41A-of enolase are both active, but have altered selectivity for divalent cations. G37A, when assayed with Mg(2+), has 12% the activity of the wild type. However, it is twice as active as wild type when assayed with Mn(2+), Zn(2+), or Co(2+). G41A has 4% the activity of the wild type with Mg(2+), is more active than wild type with Co(2+), and slightly less active than wild type with Mn(2+) and Zn(2+). The kinetic isotope effect for both mutants is greater than that of the wild type with all 4 divalent cations. These results indicate that the flexibility of this loop has subtle effects on catalytic activity.

Cloning, expression and mutagenesis of a subunit contact of rabbit muscle-specific (betabeta) enolase.

The cDNA for rabbit muscle-specific (betabeta) enolase was cloned, sequenced and expressed in Escherichia coli. This betabeta-enolase differs at eight positions from that sequenced by Chin (17). Site-directed mutagenesis was used to change residue 414 from glutamate to leucine, thereby abolishing a salt bridge involved in subunit contacts. Recombinant wild-type and mutant enolase were purified from E. coli and compared to enolase isolated from rabbit muscle. Molecular weights were determined by mass spectrometry. All three betabeta-enolases had similar kinetics, and UV and circular dichroism (CD) spectra. The mutant enolase was far more sensitive to inactivation by pressure, by KCl or EDTA, and by sodium perchlorate. We confirmed, by analytical ultracentrifugation, that the sodium perchlorate inactivation was due to dissociation. DeltaG(o) for dissociation of enolase was decreased from 49.7 kJ/mol for the wild-type enzyme to 42.3 kJ/mol for the mutant. In contrast to the wild-type enzyme, perchlorate inactivation of E414L was accompanied by a small loss of secondary structure.