title: Preparation by site-directed mutagenesis and characterization of the E211Q mutant of yeast enolase 1.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602-7229, USA.
authors.forename: V S
authors.initials: VS
authors.lastname: Sangadala
authors.forename: C V
authors.initials: CV
authors.lastname: Glover
authors.forename: R L
authors.initials: RL
authors.lastname: Robson
authors.forename: M J
authors.initials: MJ
authors.lastname: Holland
authors.forename: L
authors.initials: L
authors.lastname: Lebioda
authors.forename: J M
authors.initials: JM
authors.lastname: Brewer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010751:Phosphopyruvate Hydratase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
chemical_list: D008024:Ligands; D010751:Phosphopyruvate Hydratase