Regulation of the TCA and Glyoxylate Cycles in Brevibacterium flavum

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Abstract
Phosphoenolpyruvate carboxylase [EC 4.1.1.31] and pyruvate kinase [EC 2. 7. 1.40] were sonically extracted from Brevibacterium flavum No. 2247, and purified to the extent of 16-fold and 30-fold, respectively, by ammonium sulfate fractionation and DEAE-cellulose column chromatography. Phosphoenolpyruvate carboxylase showed an optimum pH at 6.5, required Mg2³ or Mn2³, and was activated by acetyl-coenzyme A and fructose 1, 6-diphosphate. The reaction rate-phosphoenolpyruvate concentration curve was sigmoid (Hill coefficient n=1.95), and the half maximal activity was attained at 3mM phosphoenolpyruvate. In the presence of acetyl-coenzyme A or fructose 1, 6-diphosphate the curve was hyperbolic. The enzyme was specifically inhibited by L-aspartate. The curves for the reaction rate against acetyl-coenzyme A and L-aspartate were also sigmoid in the presence of L-aspar-tate and acetyl-coenzyme A. Pyruvate kinase showed an optimum pH at 6.5 and required Mg2³ or Mn2³, but the effect of Mg2³ was very slight. Lineweaver-Burk plots for either phosphoenolpyruvate or ADP as the variable substrate at the various fixed concentrations of ADP or phoshoenolpyruvate, respectively, were parallel, suggesting that the pyruvate kinase reaction takes place according to a ping-pong mechanism. The enzyme is activated by AMP but not by acetyl-coenzyme A, fructose 1, 6-diphosphate and KC1. The rate-phosphoenolpyruvate concentration curve is sigmoid either in the presence or absence of AMP (Hill coefficient n was 2.0 or 3.0, respectively). Half maximal activity is attained at 0.25 mM phosphoenolpyruvate in the presence of 1 mM ADP. The curve for the reaction rate against ADP concentration is hyperbolic. ATP strongly inhibits the enzyme and this inhibition is non-competitive with respect to ADP. From these results, a regulation mechanism at a branching point of the glucose metabolism, phosphoenolpyruvate, is proposed.

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