REGULATION OF YEAST CTP SYNTHETASE-ACTIVITY BY PROTEIN-KINASE-C

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an al losterically regulated enzyme in the yeast Saccharomyces cerevisiae. I n 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 l evels of the PKC1 gene indicated that phosphorylation of CTP synthetas e was mediated by Pkc1p in vivo. In vitro, Pkc1p phosphorylated purifi ed CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of this acti vation involved an increase in the apparent V-max 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 k inetic behavior with respect to UTP and ATP. Phosphorylation of CTP sy nthetase did not have a significant effect on the kinetic properties o f the enzyme with respect to glutamine and GTP, Phosphorylation of CTP synthetase resulted in a decrease in the enzyme's sensitivity to prod uct inhibition by CTP. Phosphorylation did not affect the mechanism by which CTP inhibits CTP synthetase activity.