In this work we examined the regulation of CTP synthetase activity by Saccharomyces cerevisiae protein kinase C (Pkc1p) phosphorylation. Results of labeling experiments with S. cerevisiae mutants expressing different levels of the PKC1 gene indicated that CTP synthetase was phosphorylated by Pkdp in vivo. In vitro, Pkclp phosphorylated native CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of enzyme activation involved an increase in the Vmw of the reaction and an increase in the enzyme's affinity for ATP. Phosphorylated CTP synthetase also exhibited a decrease in its positive cooperative kinetic behavior with respect to UTP and ATP when compared with the native enzyme. Dephosphorylation of native CTP synthetase with alkaline phosphatase inactivated CTP synthetase activity and prevented the nucleotide-dependent tetramerization of the enzyme. Rephosphorylation of the dephosphorylated enzyme with Pkdp activated CTP synthetase activity and restored the property of nucleotide-dependent tetramerization. These results indicated that Pkdp phosphorylation of CTP synthetase regulated the enzyme by playing a role in nucleotide-dependent tetramerization and enzyme activation.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Molecular Biology