The Control of Pyruvate Kinases of Escherichia coli

Abstract

Abstract The pyruvate kinase of Escherichia coli activated by fructose 1,6-diphosphate has been purified to homogeneity. It is a tetramer of molecular weight about 240,000. By electrophoresis on sodium dodecyl sulfate polyacrylamide gels, Sephadex gel filtration in denaturing solvents, and ultracentrifugational experiments, the monomer molecular weight is found to be 60,000. Tryptophan is absent from the enzyme and no free NH2 terminus can be found. Of several nucleoside diphosphates which act as phosphate acceptors in the reaction, GDP, judging by its Km value of only 0.05 mm, is the best substrate for the enzyme. Magnesium or manganese is required as a divalent cation, but the kinetics of activation by fructose 1,6-diphosphate is different, depending upon which cation is used in assay mixtures. The enzyme is inhibited by GTP at concentrations much lower than any other nucleoside triphosphate, especially when GDP is used as the substrate. Succinyl-CoA by itself inhibits the enzyme to a small extent, but in the presence of ATP it exerts a cooperative inhibition.