Preparation by site-directed mutagenesis and characterization of the E211Q mutant of yeast enolase 1.

The published 'charge shuttle' mechanism of enolase (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822) assigns Glu-211 the task of orienting a water molecule that serves as the catalytic base which removes the proton from carbon-2 of the substrate. We prepared the E211Q mutant of yeast enolase 1 by site-directed mutagenesis. It appears to be folded correctly and to respond similarly to many of the normal ligands of enolase: it is stabilized against thermal denaturation by conformational Mg2+ and by Mg2+ and substrate and binds the chromophoric substrate analogue D-tartronate semialdehyde-2-phosphate (TSP) with affinity comparable to that of the native enzyme. However, it has only 0.01% (10(-4)) of the activity of native enolase under standard assay conditions and does not exhibit significantly more activity at various pH values or higher concentrations of substrate and Mg2+. Its ability to produce the form of enzyme-bound and reacted TSP that absorbs at shorter wavelengths is greatly slowed, while the longer wavelength absorbing form is produced rapidly. Overall, these observations are consistent with the hypothetical mechanism.