| Abstract
| - High-resolution crystallographic data show that Glu 168 and Glu211 lie on opposite surfacesof the active site from Lys 345. Two different proposals forgeneral base catalysis have emerged fromthese structural studies. In one scheme, the carboxylate sidechains of Glu 168 and Glu 211 are proposedto ionize a trapped water molecule and the OH- serves asthe base [Lebioda, L., & Stec, B. (1991)Biochemistry30, 2817−2822]. In the otherproposal, the ε-amino group of Lys 345 functions ingeneralbase catalysis [Wedekind, J. E., Poyner, R. R., Reed, G. H., &Rayment, I. (1994) Biochemistry33,9333−9342]. Genes encoding site specific mutations ofthese active site residues of yeast enolase, K345A,E168Q, and E211Q, have been prepared. The respective proteinproducts of the wild type and mutantgenes were expressed in Escherichia coli and isolated inhomogeneous form. All three mutant proteinspossess severely depressed activities in the overall reaction< 1part in 105 of wild type activity.Propertiesof the three mutant proteins in partial reactions were examined todefine more clearly the roles of theseresidues in the catalytic cycle. The K345A variant fails tocatalyze the exchange of the C-2 proton of2-phospho-d-glycerate with deuterium in D2O,whereas both the E211Q and E168Q mutant proteins arefunctional in this partial reaction. For E211Q and E168Q enolases,exchange is essentially completeprior to appearance of product, and this observation provides furthersupport for an intermediate in thenormal reaction. K345A enolase is inactive in the ionization oftartronate semialdehyde phosphate (TSP),whereas both E168Q and E211Q proteins alter the tautomeric state orcatalyze ionization of bound TSP.Wild type enolase catalyzes hydrolysis of(Z)-3-chloro-2-phosphoenolpyruvate by addition ofOH- andelimination of Cl- at C-3. This reaction mimics theaddition of OH- to C-3 of phosphoenolpyruvateinthe reverse reaction with the normal product. All three mutantproteins are depressed in their abilities tocarry out this reaction. In single-turnover assays, the activitiesvary in the order K345A > E168Q ≫E211Q. These results suggest that Lys 345 functions as the base inthe ionization of 2-PGA and that Glu211 participates in the second step of the reaction.
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