45] Phosphoenolpyruvate carboxylase from Escherichia coli

Abstract

Publisher Summary The chapter describes the purification procedure and properties of phosphoenolpyruvate carboxylase. The method is used for the purification of the enzyme from E. coli strain W, grown aerobically at 30° on a medium containing 50 mM glycerol as carbon source. Cells are harvested toward the end of the logarithmic growth phase, at densities of 0.6-0.75mg dry weight, per milliliter. The enzyme is specific for phosphoenolpyruvate, and catalyzes its carboxylation over a wide range of pH, with maximal activity at pH 8.5. The enzymatic reaction requires the presence of divalent metal ions (Mg ++ ≥Mn ++ ≥ Co ++ ). The formation of oxaloacetate from the carboxylation of phosphoenolpyruvate (PEP) is measured by coupling its reduction to malate with concomitant oxidation of reduced nicotinamide adenine dinucleotide [NADH 2 ]. This is measured as the rate of change in extinction at 340 mμ.