Reaction mechanism of phosphoenolpyruvate carboxylase. Bicarbonate-dependent dephosphorylation of phosphoenol-alpha-ketobutyrate.
Phosphoenolpyruvate carboxylase (EC 4.1.1.31) of Escherichia coli was found to catalyze the cleavage reaction of phosphoenol-alpha-ketobutyrate, a potent competitive inhibitor with the substrate, to yield inorganic phosphate and alpha-ketobutyrate. The rate of phosphate liberation was about 1/20 th of that in the normal reaction with phosphoenolpyruvate. Although HCO3- and Mg2+ were the necessary components in this reaction as in the normal reaction, no CO2 fixation could be detected. When the reaction was carried out in the presence of [18O]HCO3-, multiple incorporations of 18O atoms into the liberated phosphate molecule were observed. The molar proportions of phosphate having one, two, and three 18O atoms were 70, 25, and 5%, respectively. No multiple but only one 18O atom incorporation was observed when phosphoenolpyruvate was used as a substrate. These results suggest that the liberation of phosphate can proceed without CO2 fixation, being not consistent with the concerted mechanism [ Maruyama , H., Easterday , R. L., Chang, H. C., & Lane, M. D. (1966) J. Biol. Chem. 241, 2405-2412] but essentially consistent with the current stepwise mechanism [O'Leary, M. H., Rife , J. E., & Slater , J. D. (1981) Biochemistry 20, 7308-7314].
D001639:Bicarbonates / Q000494:pharmacology*; D002262:Carboxy-Lyases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D013058:Mass Spectrometry; D010710:Phosphates / Q000032:analysis; D010728:Phosphoenolpyruvate / Q000031:analogs & derivatives* / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D001639:Bicarbonates; D010710:Phosphates; C015443:phosphoenol-2-ketobutyrate; D010728:Phosphoenolpyruvate; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase