Octameric enolase from the hyperthermophilic bacterium Thermotoga maritima: purification, characterization, and image processing.
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+.
Schurig
H
H
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Rutkat
K
K
Rachel
R
R
Jaenicke
R
R
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013696:Temperature
D010751:Phosphopyruvate Hydratase