NUCLEOTIDE-DEPENDENT TETRAMERIZATION OF CTP SYNTHETASE FROM SACCHAROMYCES-CEREVISIAE

The nucleotide-dependent tetramerization of purified native URA7-encod ed CTP synthetase (EC 6.3,4.2, UTP: ammonia ligase (ADP-forming)) from the yeast Saccharomyces cerevisiae was characterized. CTP synthetase existed as a dimer in the absence of ATP and UTP. In the presence of s aturating concentrations of ATP and UTP, the CTP synthetase protein ex isted as a tetramer. Increasing concentrations of ATP and UTP caused a dose-dependent conversion of the dimeric species to a tetramer, The k inetics of enzyme tetramerization correlates with the kinetics of enzy me activity. The tetramerization of CTP synthetase was dependent on UT P and Mg2+ ions. ATP facilitated the UTP-dependent tetramerization of CTP synthetase by a mechanism that involved the ATP-dependent phosphor ylation of UTP catalyzed by the enzyme. The glutaminase reaction that is catalyzed by the enzyme was not required for enzyme tetramerization . CTP, a potent inhibitor of CTP synthetase activity, did not inhibit the ATP/UTP-dependent tetramerization of the enzyme. Phosphorylation o f the purified native CTP synthetase with protein kinase A and protein kinase C facilitated the nucleotide-dependent tetramerization. Dephos phorylation of native CTP synthetase with alkaline phosphatase prevent ed the nucleotide-dependent tetramerization of the enzyme. This correl ated with the inactivation of CTP synthetase activity, Rephosphorylati on of the dephosphorylated enzyme with protein kinase A and protein ki nase C resulted in a partial restoration of the nucleotide-dependent t etramerization of the enzyme. This tetramerization correlated with the partial restoration of CTP synthetase activity. Taken together, these results indicated that enzyme tetramerization was required for CTP sy nthetase activity and that enzyme phosphorylation played an important role in the tetramerization and regulation of the enzyme.