Abstract
It has long been appreciated that CTP synthetase is an allosteric enzyme that is inhibited by its endproduct, CTP. The isolation and characterization of mutant murine T lymphblasts with a genetically altered CTP synthetase activity which is refractory to inhibition by CTP have allowed an in situ determination of the essential regulatory factors governing pyrimidine synthesis and pyrimdine pool balance in a mammalian cell line. Metabolic flux and nucleotide pool measurements in unperturbed cycling cells and in cells in which the cellular pools have been exogenously manipulated indicated that the regulation of flux through CTP synthetase in situ occurs mainly by allosteric inhibition by CTP. Metabolic flux through CTP synthetase was arrested by imperceptible perturbations in CTP pools in wildtype cells but not in the mutant cells. Small changes in the levels of either ATP or GTP, a substrate and activator of the enzyme, respectively, had little influence on the in situ activity of CTP synthetase. The rates of pyrimidine synthesis are regulated in a similar fashion by uridylate nucleotides in wildtype but not in mutant cells. Thus, uridylate nucleotides govern the rates of de novo pyrimidine synthesis, while CTP modulates the balance between uridylate and cytidylate nucleotides.
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