Properties and regulation of phosphopyruvate carboxylase activity in Escherichia coli

A procedure is described for the purification of phosphopyruvate (PEP) carboxylase (EC 4.1.1.31) from extracts of Escherichia coli, strain W. The enzyme, purified 160-fold, catalysed the stoicheiometric formation of oxaloacetate from phosphopyruvate and potassium bicarbonate. This reaction occurred most readily at pH 8·5, and required the presence of divalent metal ions (Mg2+ ≥ Mn2+ > Co2+); the Km for Mg2+ was 9·8 x 10-4M. Addition of acetylcoenzyme A to the reaction system greatly stimulated the rate of oxaloacetate formation. At saturating concentrations of acetyl-coenzyme A (about 1 mM), the reaction rate was thirty times more rapid than that observed if the thiol-ester was omitted; other acyl-coenzyme A derivatives were less effective in stimulating PEP-carboxylase activity (acetyl- > propionyl- > butyryl- ≫ acrylyl- > crotonyl-coenzyme A). The Km for acetyl-coenzyme A was 1·4 x 10-4M. The effect of acetyl-coenzyme A was catalytic, and increased the apparent affinity of the enzyme for PEP from Km = 5.5 x 10-3 M to Km = 6·4 x 10-4M. Since 1 M-urea inhibited the acetyl-coenzyme A -stimulated carboxylation of PEP but did not affect the enzymic activity in the absence of acetyl-coenzyme A, it is suggested that acetyl-coenzyme A exerts its stimulatory effect through interaction with an allosteric site on the enzyme. The results obtained also suggest a mechanism for the physiological regulation of PEP -carboxylase activity in E. coli.