Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui.
D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.
Hecht
K
K
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg.
Langer
T
T
Wrba
A
A
Jaenicke
R
R
D001105:Archaea / Q000187:drug effects / Q000201:enzymology*; D001419:Bacteria / Q000201:enzymology*; D004795:Enzyme Stability; D006217:Halobacterium / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification*; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D012492:Salts / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013696:Temperature
D012492:Salts; D007770:L-Lactate Dehydrogenase