PHOSPHORYLATION AND REGULATION OF CTP SYNTHETASE FROM SACCHAROMYCES-CEREVISIAE BY PROTEIN-KINASE-A

The phosphorylation and regulation of the URA7-encoded CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cere visiae by cAMP-dependent protein kinase (protein kinase A) were examin ed. Protein kinase A is the principal mediator of signals transmitted through the RAS/cAMP pathway in S. cerevisiae. The results of labeling experiments indicated that the phosphorylation of CTP synthetase was mediated by the RAS/cAMP pathway in vivo. In vitro, protein kinase A p hosphorylated CTP synthetase at a serine residue with a stoichiometry consistent with one phosphorylation site per CTP synthetase subunit. P rotein kinase A activity was dose- and time-dependent using CTP synthe tase as a substrate. The dependence of protein kinase A activity on CT P synthetase was cooperative (n = 1.8) and the K-m value for CTP synth etase was 73 nM. Phosphorylation of CTP synthetase with protein kinase A resulted in the stimulation (190%) of activity. The mechanism of th is stimulation included an increase in the V-max of the reaction with respect to UTP and ATP, a decrease in the K-m for ATP, and a decrease in the cooperative kinetic behavior of the enzyme. Phosphorylated CTP synthetase was less sensitive to product inhibition by CTP, Protein ki nase C also phosphorylates and activates CTP synthetase. Phosphorylati on of CTP synthetase with protein kinases A and C together resulted in an increase in CTP synthetase activity that was slightly greater than that obtained when the enzyme was phosphorylated with either protein kinase alone.