Octameric enolase from the hyperthermophilic bacterium Thermotoga maritima: purification, characterization, and image processing

Protein Sci. 1995 Feb;4(2):228-36. doi: 10.1002/pro.5560040209.

Abstract

Enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) from the hyperthermophilic bacterium Thermotoga maritima was purified to homogeneity. The N-terminal 25 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources. As shown by sedimentation analysis and gel-permeation chromatography, the enzyme is a 345-kDa homoctamer with a subunit molecular mass of 48 +/- 5 kDa. Electron microscopy and image processing yield ring-shaped particles with a diameter of 17 nm and fourfold symmetry. Averaging of the aligned particles proves the enzyme to be a tetramer of dimers. The enzyme requires divalent cations in the activity assay, Mg2+ being most effective. The optimum temperature for catalysis is 90 degrees C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol-1 and 43 kJ mol-1 at temperatures below and above 45 degrees C. The pH optimum of the enzyme lies between 7 and 8. The apparent Km values for 2-phospho-D-glycerate and Mg2+ at 75 degrees C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors 2 and 30, respectively. Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 degrees C in the absence and in the presence of Mg2+.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Enzyme Stability
  • Gram-Negative Anaerobic Bacteria / enzymology*
  • Gram-Negative Anaerobic Bacteria / growth & development
  • Hydrogen-Ion Concentration
  • Microscopy, Electron
  • Molecular Sequence Data
  • Molecular Weight
  • Phosphopyruvate Hydratase / chemistry*
  • Phosphopyruvate Hydratase / isolation & purification
  • Phosphopyruvate Hydratase / metabolism
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship
  • Temperature

Substances

  • Phosphopyruvate Hydratase