Purification and properties of pyruvate kinase from Streptococcus sanguis and activator specificity of pyruvate kinase from oral streptococci.
It was found that pyruvate kinases with two different regulatory characteristics were distributed among oral streptococci. The pyruvate kinases of Streptococcus mutans, Streptococcus salivarius, and Streptococcus bovis were activated by glucose 6-phosphate, whereas the enzymes of both Streptococcus sanguis and Streptococcus mitis were activated by fructose 1,6-bisphosphate. Pyruvate kinase (EC 2.7.1.40) from S. sanguis NCTC 10904 was purified, giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 250,000 to 260,000 and consisted of four identical subunits. Whereas the pyruvate kinase from S. mutans was completely dependent on glucose 6-phosphate (K. Abbe and T. Yamada, J. Bacteriol. 149:299-305, 1982), the enzyme from S. sanguis was activated by fructose 1,6-bisphosphate. In the presence of 0.5 mM fructose 1,6-bisphosphate, the saturation curves for the substrates, phosphoenolpyruvate and ADP, were hyperbolic, and the Km values were 0.13 and 0.30 mM, respectively. Without fructose 1,6-bisphosphate, however, saturation curves for both substrates were sigmoidal. GDP, IDP, and UDP could replace ADP. Like the enzyme from S. mutans, the enzyme from S. sanguis required a divalent cation, Mg2+ or Mn2+, and a monovalent cation, K+ or NH4+, for activity, and it was strongly inhibited by Pi. When the concentration of Pi was increased, the half-saturating concentration and Hill coefficient for fructose 1,6-bisphosphate increased. The remarkable fluctuation of intracellular levels of fructose 1,6-bisphosphate and phosphoenolpyruvate observed in the cells growing under glucose limitation and nitrogen limitation implies that the intracellular concentration of fructose 1,6-bisphosphate, in cooperation with that of Pi, may regulate pyruvate kinase activity in S. sanguis in vivo.
D002412:Cations / Q000494:pharmacology; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000494:pharmacology*; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology*; D006019:Glycolysis; D006598:Hexosediphosphates / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D013045:Species Specificity; D013291:Streptococcus / Q000201:enzymology*; D013298:Streptococcus sanguis / Q000201:enzymology*
D002412:Cations; D005635:Fructosediphosphates; D005958:Glucosephosphates; D006598:Hexosediphosphates; D010710:Phosphates; D019298:Glucose-6-Phosphate; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate