Characterization of a novel dUTP-dependent activity of CTP synthetase from Saccharomyces cerevisiae.
CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzyme concentration. Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0. The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively. dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity. dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity. The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP. The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations. These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant.
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Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901, USA.
Park
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Carman
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D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003570:Cytidine Triphosphate / Q000737:chemistry; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000737:chemistry*; D019281:Dimerization; D004789:Enzyme Activation; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D003845:Deoxycytosine Nucleotides; D003856:Deoxyuracil Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D003570:Cytidine Triphosphate; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase