Reaction mechanism of phosphoenolpyruvate carboxylase. Bicarbonate-dependent dephosphorylation of phosphoenol-.alpha.-ketobutyrate

Abstract

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) of Escherichia coli catalyzed the cleavage reaction of phosphoenol-.alpha.-ketobutyrate, a potent competitive inhibitor with the substrate, to yield Pi and .alpha.-ketobutyrate. The rate of phosphate liberation was about 1/20 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 1, 2 and 3 18O atoms were 70, 25 and 5%, respectively. No multiple but only 1 18O atom incorporation was observed when phosphoenolpyruvate was used as a substrate. Thus, the liberation of phosphate can proceed without CO2 fixation, being not consistent with the concerted mechanism but essentially consistent with the current stepwise mechanism.