Regulation of the TCA and Glyoxylate Cycles in Brevibacterium flavum

II. Regulation of Phosphoenolpyruvate Carboxylase and Pyruvate Kinase

Abstract

Phosphoenolpyruvate carboxylase [EC 4. 1. 1. 31] and pyruvate kinase [EC 2. 7. 1. 40] were sonically extracted from Brevibacterium fiavum 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 Mg⁺ or Mn²⁺, 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 3 mm 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 Mg²⁺ or Mn²⁺, but the effect of Mg²⁺ was very slight. Lineweaver-Burk plots for either phosphoenolpyruvate or ADP as the variable substrate at the various fixed concentrations of ADP or phos-phoenolpyruvate, 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 KCl. The rate-phospho-enolpyruvate 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.