The synergistic inhibition of Escherichia coli aspartate carbamoyltransferase by UTP in the presence of CTP is due to the binding of UTP to the low affinity CTP sites.
Escherichia coli aspartate carbamoyltransferase controls pyrimidine biosynthesis by feedback inhibition involving both CTP and UTP, although UTP only inhibits the enzyme in the presence of CTP (Wild, J. R., Loughrey-Chen, S. J., and Corder, T. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 46-50). The mechanism by which the enzyme can discriminate between these two pyrimidines is unknown, as well as where UTP binds and its mode of action. A mutant version of the enzyme with a single amino acid substitution in the regulatory site (Asp-19----Ala) causes loss of the synergistic inhibition of UTP in the presence of CTP, and furthermore, this enzyme is inhibited by UTP alone. Analysis of CTP binding to the mutant enzyme reveals that UTP can bind to the mutant enzyme in the absence of CTP but not in its presence. This is completely opposite to the wild-type enzyme in which case UTP only exhibits significant binding in the presence of CTP. Further analysis of the binding data for the wild-type enzyme reveals that, in the presence of UTP, CTP only binds to three sites, although CTP binds to six sites, three with high affinity and three with low affinity in the absence of UTP. Parallel UTP binding experiments in the presence of CTP suggest that UTP binds to the three weak CTP sites. The Asp-19----Ala substitution prevents UTP binding in the presence of CTP and allows UTP to bind and inhibit the enzyme in the absence of CTP. Since the x-ray data indicate no specific interactions between the amino group of cytosine and amino acid side chains in the regulatory binding site, the discrimination between UTP and CTP by the wild-type enzyme must be due to subtle differences in the binding sites rather than direct side chain contacts.
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167.
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Zhang
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Kantrowitz
D000409:Alanine; D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics; D001224:Aspartic Acid; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D004357:Drug Synergism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D014544:Uridine Triphosphate / Q000378:metabolism / Q000494:pharmacology*
D001224:Aspartic Acid; D003570:Cytidine Triphosphate; D001221:Aspartate Carbamoyltransferase; D000409:Alanine; D014544:Uridine Triphosphate