We have previously reported that phosphoenolpyruvate carboxykinase (PCK) overexpression under glycolytic conditions enables Escherichia coli to harbor a high intracellular ATP pool resulting in enhanced recombinant protein synthesis. To estimate how much PCK-mediated phosphoenolpyruvate (PEP) carboxylation is contributed to the ATP increase under engineered conditions, the kinetics of PEP carboxylation by PCK and substrate competing phosphoenolpyruvate carboxylase (PPC) were measured using recombinant enzymes. The PEP carboxylation catalytic efficiency (k(cat)/K-m) of the recombinant PCK was 660 mM(-1) min(-1), whereas that of the recombinant PPC was 1500 mM(-1) min(-1). Under the presence of known allosteric effectors (fructose 1,6-bisphosphate, acetyl-CoA, ATP, malate, and aspartate) close to in vivo conditions, the catalytic efficiency of PCK-mediated PEP carboxylation (84 mM(-1) min(-1)) was 28-folds lower than that of PPC (2370 mM(-1) min(-1)). To verify the above results, an E. coil strain expressing native PCK and PPC under control of identical promoter was constructed by replacing PCK promoter region with that of PPC in chromosome. The native PCK activity (33 nmol/mg(-protein) min) was 5-folds lower than PPC activity (160 nmol/mg(-protein) min) in the cell extract from the promoter-exchanged strain. Intracellular modifications of ATP concentration by PCK activity and the consequences for biotechnology are further discussed. (C) 2013 Elsevier Inc. All rights reserved.