Activation of phosphoenolpyruvate carboxykinase isolated from Veillonella parvula.

Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) has been purified 940-fold from Veillonella parvula using protamine sulphate treatment, ammonium sulphate precipitation, and column chromatography. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity in the direction of oxaloacetate (OAA) decarboxylation was exhibited at pH 9.0. At this pH, the V. parvula enzyme catalysed phosphoenolpyruvate formation in the presence of Mn2+ ions. In the presence of varying concentrations of OAA and ATP, the PEPCK from V. parvula exhibited hyperbolic kinetics with KmS of 0.16 and 0.46 mM, respectively. PEPCK from the anaerobe was not inhibited by NADH, succinate, glutamate, D-glucose-6-phosphate, acetyl phosphate, D-fructose, 1,6-bisphosphate, pyruvate, ribose 5-phosphate, and aspartate. However, acetyl CoA, glyceraldehyde 3-phosphate, 3-phospho-D-glycerate, CTP, and GTP activated the enzyme. The activation of acetyl CoA was uncompetitive and noncooperative.