title: Synergistic inhibition of Escherichia coli aspartate transcarbamylase by CTP and UTP: binding studies using continuous-flow dialysis.
abstract: The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves feedback inhibition by both CTP and UTP, although it is only in the presence of CTP that UTP appears to inhibit the activity of the enzyme. In order to better understand the parts played by both pyrimidine nucleotides in this synergistic inhibition, binding studies were performed by continuous-flow dialysis and ultracentrifugation methods. The results obtained show that UTP binds to ATCase in the absence of CTP. Nevertheless, this binding does not induce any inhibition unless CTP is present. The mutual influence of CTP and UTP on their respective binding constants suggests that they bind to the same regulatory sites. However, the results obtained cannot be satisfactorily explained by a simple competition between the nucleotides, and it is shown that reciprocal affinity enhancements play a fundamental role. CTP enhances the affinity of UTP for the regulatory sites 80-fold, and conversely, UTP enhances the affinity of CTP 5-fold. Interestingly, the isolated regulatory subunits bind the two pyrimidine nucleotides following the same pattern as the entire enzyme. These observations indicate that the synergistic inhibition mechanism relies entirely on interactions between the two adjacent allosteric sites which belong to the same regulatory dimer.
authors.affiliation: Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
authors.forename: P
authors.initials: P
authors.lastname: England
authors.forename: G
authors.initials: G
authors.lastname: Hervé
mesh_terms: D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001667:Binding, Competitive; D003570:Cytidine Triphosphate / Q000378:metabolism*; D003956:Dialysis; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D014461:Ultracentrifugation; D014544:Uridine Triphosphate / Q000378:metabolism*
chemical_list: D008024:Ligands; D003570:Cytidine Triphosphate; D001221:Aspartate Carbamoyltransferase; D014544:Uridine Triphosphate