Phosphoenolpyruvate Carboxylase of Escherichia coil. Inhibition by Various Analogs and Homologs of Phosphoenolpyruvate1

Abstract

In an attempt to investigate the topography of the catalytic site of phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli, the inhibitor constants (Ki) for more than 20 compounds were determined with the reaction system containing dioxane, a non-physiological activator of the enzyme. The Ki values for the compounds lacking methylene-, carboxylate-, or phosphate groups were all more than 10-fold larger than the Km value for PEP, indicating the significant contribution of these groups to the binding of PEP with the enzyme. The Ki value for L-phospholactate (0.30 mM) was almost equal to the Km value for PEP (0.25 mM), whereas that for D-phospholactate (0.89 mM) was about 3-fold larger than the Km value. It was presumed that PEP binds with the enzyme on its si-side. Among 6 PEP homologs, the Ki values for phosphoenol alpha-ketobutyrate (0.024 mM) and phosphoenol alpha-ketovalerate (0.034 mM) were about one-tenth the Km value, indicating the presence of a hydrophobic pocket around the binding site of the methylene group of PEP, where the carboxylation reaction is supposed to occur. DL-Phosphomalate, a presumptive carboxylated substrate, was a weak inhibitor with a Ki value of 2.20 mM.