Properties and regulation of phosphopyruvate carboxylase activity in Escherichia coli

Abstract

A procedure is described for the purification of phosphopyruvate (PEP) carboxylase (EC 4.1.1.31) from extracts of E. coli, strain-W. The enzyme, purified 160-fold, catalyzed 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 10-4 M. Addition of acetylcoenzyme-A to the reaction system greatly stimulated the rate of oxaloacetate formation. At saturating concentrations of acetyl-coenzyme-A (about 1mM), the reaction rate was thirty times more rapid than that observid 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 10-4 M. 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 10-4 M. 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.