Regulation of yeast CTP synthetase activity by protein kinase C.
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme in the yeast Saccharomyces cerevisiae. In this work we examined the regulation of CTP synthetase activity by S. cerevisiae protein kinase C (Pkc1p) phosphorylation. The results of labeling experiments with S. cerevisiae mutants expressing different levels of the PKC1 gene indicated that phosphorylation of CTP synthetase was mediated by Pkc1p in vivo. In vitro, Pkc1p phosphorylated purified CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of this activation involved an increase in the apparent Vmax of the reaction and an increase in the enzyme's affinity for ATP. In vitro phosphorylated CTP synthetase also exhibited a decrease in its positive cooperative kinetic behavior with respect to UTP and ATP. Phosphorylation of CTP synthetase did not have a significant effect on the kinetic properties of the enzyme with respect to glutamine and GTP. Phosphorylation of CTP synthetase resulted in a decrease in the enzyme's sensitivity to product inhibition by CTP. Phosphorylation did not affect the mechanism by which CTP inhibits CTP synthetase activity.
Yang
W L
WL
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903, USA.
Bruno
M E
ME
Carman
G M
GM
D000255:Adenosine Triphosphate / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D003570:Cytidine Triphosphate / Q000378:metabolism; D004789:Enzyme Activation; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000096:biosynthesis / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D005973:Glutamine; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase