| Abstract
| - 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-4position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as asubstrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzymeconcentration. Maximum dUTP-dependent activity was dependent on MgCl2 (4 mM) and GTP (Ka = 14μM) at a pH optimum of 8.0. The apparent Km 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 ofenzyme 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 synthesisof dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major productsynthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP werepresent at concentrations near their Km 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 concentrationnear its Km value and dUTP was varied from subsaturating to saturating concentrations. These data suggestedthat the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant.
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