Phosphoenolpyruvate Carboxylase of Escherichia coli: PURIFICATION AND SOME PROPERTIES

Abstract

Abstract Phosphoenolpyruvate carboxylase was purified from Escherichia coli, strain B (ATCC 11303), and its properties studied. Its amino acid composition and some of its structural properties closely resemble those of the enzyme recently purified by Smith ((1971) J. Biol. Chem. 246, 4234–4241). Steady state kinetics, studied in the presence of the activators acetyl coenzyme A, fructose diphosphate, guanosine triphosphate, and 2-mercaptoethanol, and the inhibitor, l-aspartate, suggest an allosteric enzyme going through ligand-dependent transitions between two discrete states. This conclusion is supported by measurements of optical rotation which show two extreme values attainable in saturating activator, and inhibitor, respectively. The active (R) state is characterized by a single value for Vmax and by a Km of about 0.6 mm for phosphoenolpyruvate, approached at infinite activator concentration. The alternate (T) state, stabilized by aspartate, is catalytically completely inert. The property which distinguishes this enzyme from other phosphoenolpyruvate carboxylases is that upon addition of substrate, steady state velocity develops after a lag period of variable duration. Substrate and activators shorten, but the inhibitor prolongs this lag period. Analysis of the dependence of the lag period on enzyme concentration suggests that at assay dilutions (7 µg per ml) the enzyme is present as an inactive dimer of two peptide chains (mol wt 100,000) which upon addition of substrate, or activators, associates to the active tetramer. The lag period does not completely vanish even at infinite enzyme, substrate, or activator concentrations. The residual lag period is associated with a modification of the enzyme by the substrate, for once the enzyme has reacted with substrate the residual lag period vanishes for several hours, and then gradually returns. A model, compatible with these observations, is suggested.