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].[1]References
- Reaction mechanism of phosphoenolpyruvate carboxylase. Bicarbonate-dependent dephosphorylation of phosphoenol-alpha-ketobutyrate. Fujita, N., Izui, K., Nishino, T., Katsuki, H. Biochemistry (1984) [Pubmed]
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