Preparation and characterization of the E168Q site-directed mutant of yeast enolase 1.
Yeast has two enolase isozymes (called 1 and 2), either of which suffices for growth. We cloned DNA encoding the enolase 1 protein coding and promoter regions flanked by BamHI termini using the PCR. The DNA, which 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 resulting plasmid-containing strain makes enolase 1 in quantities which depend on cell growth. A "charge shuttle" mechanism of action of enolase based on X-ray crystallographic evidence (Lebioda and Stec, Biochemistry 30:2817, 1991) involves Glu-168 accepting a proton from a water molecule that in turn accepts a proton from carbon-2 of the substrate. We prepared the E168Q mutant of enolase 1 by oligonucleotide-directed site-directed mutagenesis. Its identity was confirmed by N-terminal sequence analysis, HPLC on Superose 12, SDS-gel electrophoresis, and the sequence of the mutated DNA protein-coding region. The E168Q mutant has approximately 0.01% of the activity of native enolase. It binds substrate/product, AEP (3-aminoenolpyruvate-2-phosphate, the 3-amino analogue of the product phosphoenolpyruvate) and TSP (D-tartronate semialdehyde-2-phosphate, the aldehyde analogue of the substrate 2-phosphoglycerate), 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 shuttle" mechanism; the reaction with TSP, which presumably requires proton removal from carbon-2, is complex but shows a very slow phase consistent with expectations.
Brewer
J M
JM
Department of Biochemistry, University of Georgia, Athens 30602.
Robson
R L
RL
Glover
C V
CV
Holland
M J
MJ
Lebioda
L
L
D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010751:Phosphopyruvate Hydratase / Q000235:genetics* / Q000502:physiology; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D015003:Yeasts / Q000201:enzymology*
D010751:Phosphopyruvate Hydratase