Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants.
High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345. Two different proposals for general base catalysis have emerged from these structural studies. In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base [Lebioda, L., & Stec, B. (1991) Biochemistry 30, 2817-2822]. In the other proposal, the epsilon-amino group of Lys 345 functions in general base catalysis [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. Genes encoding site specific mutations of these active site residues of yeast enolase, K345A, E168Q, and E211Q, have been prepared. The respective protein products of the wild type and mutant genes were expressed in Escherichia coli and isolated in homogeneous form. All three mutant proteins possess severely depressed activities in the overall reaction- < 1 part in 10(5) of wild type activity. Properties of the three mutant proteins in partial reactions were examined to define more clearly the roles of these residues in the catalytic cycle. The K345A variant fails to catalyze the exchange of the C-2 proton of 2-phospho-D-glycerate with deuterium in D2O, whereas both the E211Q and E168Q mutant proteins are functional in this partial reaction. For E211Q and E168Q enolases, exchange is essentially complete prior to appearance of product, and this observation provides further support for an intermediate in the normal reaction. K345A enolase is inactive in the ionization of tartronate semialdehyde phosphate (TSP), whereas both E168Q and E211Q proteins alter the tautomeric state or catalyze ionization of bound TSP. Wild type enolase catalyzes hydrolysis of (Z)-3-chloro-2-phosphoenolpyruvate by addition of OH- and elimination of Cl- at C-3. This reaction mimics the addition of OH- to C-3 of phosphoenolpyruvate in the reverse reaction with the normal product. All three mutant proteins are depressed in their abilities to carry out this reaction. In single-turnover assays, the activities vary in the order K345A > E168Q >> E211Q. These results suggest that Lys 345 functions as the base in the ionization of 2-PGA and that Glu 211 participates in the second step of the reaction.
Poyner
R R
RR
Institute for Enzyme Research, Graduate School, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53705, USA.
Laughlin
L T
LT
Sowa
G A
GA
Reed
G H
GH
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D005988:Glyceric Acids / Q000378:metabolism; D006868:Hydrolysis; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000031:analogs & derivatives / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013646:Tartronates / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology / Q000235:genetics
D005988:Glyceric Acids; D011994:Recombinant Proteins; D013646:Tartronates; C059689:tartronate semialdehyde phosphate; C063016:phosphoenol-3-chloropyruvate; C008885:2-phosphoglycerate; D010728:Phosphoenolpyruvate; D010751:Phosphopyruvate Hydratase