Enzymatic function of loop movement in enolase: preparation and some properties of H159N, H159A, H159F, and N207A enolases.
The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases. These have 0.07%-0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered. Activation by Mg2+ is biphasic, with the smaller Mg2+ activation constant closer to that of the "catalytic" Mg2+ binding site of native enolase and the larger in the mM range in which native enolase is inhibited. A third Mg2+ may bind to the phosphoryl, functionally replacing proton donation by His159. N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds. It has 21% of the native activity, also exhibits biphasic Mg2+ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction. Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.
D000818:Animals; D002152:Calorimetry, Differential Scanning; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013646:Tartronates / Q000494:pharmacology; D013696:Temperature
D021122:Protein Subunits; D013646:Tartronates; C059689:tartronate semialdehyde phosphate; D006639:Histidine; D010751:Phosphopyruvate Hydratase; D008274:Magnesium