Purification and properties of pyruvate kinase from Bacillus stearothermophilus.
Pyruvate kinase was purified to homogeneity from a moderate thermophile, Bacillus stearothermophilus. The molecular weight of the enzyme was found to be 250,000 on gel filtration and 242,000 on sedimentation analysis. The enzyme consisted of four identical subunits of a molecular weight of 62,000-64,000. There were no remarkable differences between the thermophilic enzyme and mesophilic enzymes in amino acid composition, secondary structure, mono- and di-valent cation requirements for activity or specificity for nucleoside diphosphates. But the thermophilic enzyme was stable at high temperature and for a longer period of storage at lower temperature. Its specific activity was relatively high even at a low temperature (30 degrees C). The enzyme exhibited homotropic positive cooperativity for phosphoenol-pyruvate, but not for ADP. It was allosterically activated by AMP, ribose 5-phosphate and nucleoside monophosphates, but not by fructose 1,6-bisphosphate. Activation by AMP and ribose 5-phosphate, and inhibition by inorganic phosphate were also observed even at the physiological temperature (60 degrees C) for the thermophile.
Sakai
H
H
Suzuki
K
K
Imahori
K
K
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000302:isolation & purification*; D012267:Ribosemonophosphates / Q000494:pharmacology; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D002413:Cations, Divalent; D002414:Cations, Monovalent; D010710:Phosphates; D012267:Ribosemonophosphates; D000249:Adenosine Monophosphate; C031626:ribose-5-phosphate; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D008274:Magnesium