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 S tec, B. (1991) Biochemistry 30, 2817-2822) assigns Glu-211 the task of orienting a water molecule that serves as the catalytic base which re moves 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 li gands of enolase: it is stabilized against thermal denaturation by con formational Mg2+ and by Mg2+ and substrate and binds the chromophoric substrate analogue D-tartronate semialdehyde-2-phosphate (TSP) with af finity comparable to that of the native enzyme. However, it has only 0 .01% (10(-4)) of the activity of native enolase under standard assay c onditions and does not exhibit significantly more activity at various pH values or higher concentrations of substrate and Mg2+ Its ability t o produce the form of enzyme-bound and reacted TSP that absorbs at sho rter wavelengths is greatly slowed, while the longer wavelength absorb ing form is produced rapidly. Overall, these observations are consiste nt with the hypothetical mechanism.