Evidence for two activated forms of pyruvate kinase from Bacillus stearothermophilus in the presence of ribose 5-phosphate.
Allosteric activation of pyruvate kinase from a thermophilic bacterium, Bacillus stearothermophilus, by ribose 5-phosphate (R5P) was kinetically examined. Two activated forms of this enzyme could be distinguished, depending on the R5P concentration. One form (Form I) was observed at about 10(-5) M R5P. It showed a slightly negative cooperativity for phosphoenolpyruvate (PEP). The other form (Form II) was observed at more than 10(-3) M R5P and showed Michaelis-Menten kinetics for PEP. The PEP and ADP concentrations that yield half-maximal velocity were essentially identical for the two forms (about 0.1 and about 0.5 mM, respectively), but Form I had a larger Vmax value than Form II. In the absence of R5P, the enzyme showed a homotropic positive cooperativity for PEP; the concentration required for the half-maximal velocity was about 2 mM and that of ADP was about 1.6 mM. The enzyme was more susceptible to protease digestion in the presence of R5P than in the absence of it. The concentration of R5P required for the enzyme to be susceptible to protease digestion was approximately identical with that required to generate Form I. With more than 10(-3) M R5P, the thermostability of the enzyme was greatly increased. The concentration of R5P required for the enzyme to be thermostable was in good agreement with that required to generate Form II. These results indicate that the two activated forms distinguished kinetically differ in their conformations, too. The saturating level of PEP did not cause such a change in the thermostability or the susceptibility to protease.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D010428:Pentosephosphates / Q000494:pharmacology*; D010754:Phosphoribosyl Pyrophosphate / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000494:pharmacology*
D007527:Isoenzymes; D010428:Pentosephosphates; D012267:Ribosemonophosphates; C031626:ribose-5-phosphate; D000244:Adenosine Diphosphate; D010754:Phosphoribosyl Pyrophosphate; D011770:Pyruvate Kinase