Purification and properties of pyruvate kinase from Streptococcus sanguis and activator specificity of pyruvate kinase from oral streptococci

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Abstract
Pyruvate kinases with 2 different regulatory characteristics were distributed among oral streptococci. The pyruvate kinases of S. mutans, S. salivarius and S. bovis were activated by G-6-P; the enzymes of S. sanguis and S. 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 MW of 250,000-260,000 and consisted of 4 identical subunits. Whereas the pyruvate kinase from S. mutans was completely dependent on G-6-P, 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, 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; 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 N 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.

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