In situ regulation of mammalian CTP synthetase by allosteric inhibition.
The regulatory role of the allosteric site of CTP synthetase on flux through the enzyme in situ and on pyrimidine nucleotide triphosphate (NTP) pool balance was investigated using a mutant mouse T lymphoblast (S49) cell line which contains a CTP synthetase refractory to complete inhibition by CTP. Measurements of [3H]uridine incorporation into cellular pyrimidine NTP pools as a function of time indicated that CTP synthesis in intact wild type cells was markedly inhibited in a cooperative fashion by small increases in CTP pools, whereas flux across the enzyme in mutant cells was much less affected by changes in CTP levels. The cooperativity of the allosteric inhibition of the enzyme was greater in situ than in vitro. Exogenous manipulation of levels of GTP, an activator of the enzyme, indicated that GTP had a moderate effect on enzyme activity in situ, and changes in pools of ATP, a substrate of the enzyme, had small effects on CTP synthetase activity. The consequences of incubation with actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine on the flux across CTP synthetase and on NTP pools differed considerably between wild type and mutant cells. Under conditions of growth arrest, an intact binding site for CTP on CTP synthetase was required to maintain a balance between the CTP and UTP pools in wild type cells. Moreover, wild type cells failed to incorporate H14CO3- into pyrimidine pools following growth arrest. In contrast, mutant cells incorporated the radiolabel at a high rate indicating loss of a regulatory function. These results indicated that uridine nucleotides are important regulators of pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CTP pools.
D000818:Animals; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003562:Cytidine / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D008025:Ligases / Q000037:antagonists & inhibitors*; D051379:Mice; D012313:RNA / Q000378:metabolism; D014529:Uridine / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
D003562:Cytidine; D012313:RNA; D003570:Cytidine Triphosphate; D004247:DNA; D003513:Cycloheximide; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D014544:Uridine Triphosphate; D014529:Uridine