title: Purification and properties of pyruvate kinase from Streptococcus sanguis and activator specificity of pyruvate kinase from oral streptococci.
abstract: 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.
mesh_terms: 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*
chemical_list: D002412:Cations; D005635:Fructosediphosphates; D005958:Glucosephosphates; D006598:Hexosediphosphates; D010710:Phosphates; D019298:Glucose-6-Phosphate; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate