Investigation of the mechanism of CTP synthetase using rapid quench and isotope partitioning methods.
The UTP-dependent ATPase reaction and the glutamine-dependent overall reaction of Escherichia coli CTP synthetase have been studied by rapid quench and isotope partitioning kinetics. The effect of GTP, an allosteric effector, on the pre-steady-state kinetics of both reactions has also been examined. The time courses of the UTP-dependent ATPase reaction in the presence and absence of GTP are both characterized by a burst of acid-labile phosphate equivalent to 0.93 and 0.43 subunits, respectively. The time course of the glutamine-dependent reaction in the absence of GTP is also characterized by a burst of acid-labile phosphate corresponding to 0.8 subunit; however, in the presence of GTP, no burst was observed. These results along with positional isotope exchange experiments [von der Saal, W., Anderson, P. M., & Villafranca, J. J. (1985) J. Biol. Chem. 260, 14997] provide evidence that the mechanism of CTP formation involves phosphorylation of UTP followed by attack of NH3, and finally release of phosphate, producing CTP, ADP, and Pi. A kinetic model for the first stages of the enzymatic reaction was developed from the rapid quench data, and the internal equilibrium constant for the formation of the phosphorylated UTP intermediate was determined. The internal equilibrium constants for the UTP-dependent reaction in the presence and absence of GTP were found to be 1.1 and 18, respectively. By contrast, the internal equilibrium constant for the reaction in the presence of glutamine was 50. Thus, the presence of glutamine shifts the internal equilibrium constant to favor formation of the phosphorylated UTP intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)
Lewis
D A
DA
Department of Chemistry, Pennsylvania State University, University Park 16802.
Villafranca
J J
JJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D055598:Chemical Phenomena; D002621:Chemistry; D003570:Cytidine Triphosphate / Q000096:biosynthesis*; D003597:Cytosine Nucleotides / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D014544:Uridine Triphosphate / Q000378:metabolism
D003597:Cytosine Nucleotides; D010761:Phosphorus Radioisotopes; D005973:Glutamine; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D014544:Uridine Triphosphate