Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.
CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.
D000494:Allosteric Regulation; D002151:Calorimetry; D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000096:biosynthesis; D005972:Glutaminase / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology*; D008954:Models, Biological
D005973:Glutamine; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D005972:Glutaminase; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase