PREPARATION AND CHARACTERIZATION OF THE E168Q SITE-DIRECTED MUTANT OFYEAST ENOLASE-1

Yeast has two enolase isozymes (called 1 and 2), either of which suffi ces for growth. We cloned DNA encoding the enolase 1 protein coding an d promoter regions flanked by BamHI termini using the PCR. The DNA, wh ich contained no nucleotide base changes altering the protein sequence , was cloned into the multicopy shuttle vector pRS314 and transformed into a yeast strain with a deletion in its enolase 1 gene. The resulti ng plasmid-containing strain makes enolase 1 in quantities which depen d on cell growth. A ''charge shuttle'' mechanism of action of enolase based on X-ray crystallographic evidence (Lebioda and Stec, Biochemist ry 30:2817, 1991) involves Glu-168 accepting a proton from a water mol ecule that in turn accepts a proton from carbon-2 of the substrate. We prepared the E168Q mutant of enolase 1 by oligonucleotide-directed si te-directed mutagenesis. Its identity was confirmed by N-terminal sequ ence analysis, HPLC on Superose 12, SDS-gel electrophoresis, and the s equence of the mutated DNA protein-coding region. The E168Q mutant has approximately 0.01% of the activity of native enolase. It binds subst rate/product, AEP (3-aminoenolpyruvate-2-phosphate, the 3-amino analog ue of the product phosphoenolpyruvate) and TSP (D-tartronate semialdeh yde-2-phosphate, the aldehyde analogue of the substrate 2-phosphoglyce rate), the latter two at least with affinities similar to those of the native enzyme. The E168Q enolase also produces absorbance changes in the analogues. The reaction with AEP is consistent with the ''charge s huttle'' mechanism; the reaction with TSP, which presumably requires p roton removal from carbon-2, is complex but shows a very slow phase co nsistent with expectations. (C) 1993 Wiley-Liss, Inc.