Abstract
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.
Highlights
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADPforming)) is an allosterically regulated enzyme that is essential for the growth and metabolism of cells
The positive cooperative kinetics toward UTP and ATP is attributed to the nucleotide-dependent tetramerization of the enzyme [3, 15]
In S. cerevisiae, CTP synthetase is encoded by the URA7 and URA8 genes [8, 9]
Summary
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADPforming)) is an allosterically regulated enzyme that is essential for the growth and metabolism of cells. CTP synthetase catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP [2, 3]. A characteristic common to the pure CTP synthetases is the inhibition of their activities by the product CTP [3, 12,13,14]. CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP [12,13,14]. Our studies on the pure URA7-encoded enzyme have revealed that CTP synthetase is regulated by protein kinase C phosphorylation [31].
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