Enzymatic function of loop movement in enolase: preparation and some properties of H159N, H159A, H159F, and N207A enolases

J Protein Chem. 2003 May;22(4):353-61. doi: 10.1023/a:1025390123761.


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.

MeSH terms

  • Animals
  • Calorimetry, Differential Scanning
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Histidine / genetics
  • Histidine / metabolism
  • Hydrogen-Ion Concentration
  • Magnesium / pharmacology
  • Mutagenesis, Site-Directed / genetics
  • Mutation / genetics
  • Phosphopyruvate Hydratase / chemistry*
  • Phosphopyruvate Hydratase / genetics
  • Phosphopyruvate Hydratase / metabolism*
  • Protein Structure, Quaternary / drug effects
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Tartronates / pharmacology
  • Temperature


  • Protein Subunits
  • Tartronates
  • tartronate semialdehyde phosphate
  • Histidine
  • Phosphopyruvate Hydratase
  • Magnesium