Identification of critical residues in the bifunctional phosphoenolpyruvate synthetase kinase/phosphotransferase of Escherichia coli

In bacteria, phosphoenolpyruvate synthetase (EC 2.7.9.2) catalyses the conversion of pyruvate to phosphoenolpyruvate during gluconeogenesis. The enzyme is regulated by an unusual bifunctional serine/threonine kinase-phosphotransferase (PEP synthetase regulatory protein) that involves an ADP-dependent phosphorylation and a Pi-dependent dephosphorylation mechanism. Site-directed mutagenesis studies have revealed that two separate regions of Escherichia coli PEP synthetase regulatory protein are involved in catalysis; a central P-loop that is probably critical for the binding of the protein substrate (PEP synthetase) and a C-terminal region that interacts with the P-loop and is required to bind ADP and Pi. In addition, our findings are consistent with the P-loop and the C-terminal region responsible for ADP and Pi binding being juxtaposed in the functioning enzyme. Two closely associated active sites are present in E. coli PEP synthetase regulatory protein. Given the high degree of sequence similarity between bacterial PEP synthetase regulatory protein and plant pyruvate, orthophosphate dikinase regulatory protein, it is highly likely that there are two active sites involved in the ADP-dependent inactivation and the Pi-dependent activation of both PEP synthetase and pyruvate, orthophosphate dikinase and they are very close together