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

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 homooctamer 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 °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 °C. The pH optimum of the enzyme lies between 7 and 8. The apparent K_m values for 2-phospho-D-glycerate and Mg²⁺ at 75 °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 K_i of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 °C in the absence and in the presence of Mg²⁺.