Involvement of the gamma-phosphate of UTP in the synergistic inhibition of Escherichia coli aspartate transcarbamylase by CTP and UTP.
The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves synergistic feedback inhibition by CTP and UTP. Previously reported results [England, P., & Hervé, G. (1992) Biochemistry 31, 9725-9732] suggest that this phenomenon relies entirely on interactions between the two neighboring allosteric sites, which belong to the same regulatory dimer. Furthermore, it has been demonstrated that UTP alone binds to the enzyme, but that it is only in the presence of CTP that this binding inhibits the catalytic activity. The properties of mutants in which the synergistic inhibition is totally abolished suggested that the terminal gamma-phosphate of the pyrimidine triphosphate nucleotides may play a crucial role in promoting site-site interactions within the regulatory dimer. In the present work, kinetic studies and binding experiments by continuous-flow dialysis were performed, using combinations of diphosphate and triphosphate nucleotides. The results obtained show that the gamma-phosphate lf UTP is indeed essential for synergistic inhibition to occur, as UDP is unable to inhibit ATCase activity, whether alone or in combination with CTP. On the contrary, the gamma-phosphate of CTP can be suppressed without modifying the inhibitory properties of this nucleotide and its synergy of action with UTP. These results indicate that the mutual effects of CTP and UTP on their respective binding are not symmetrical and that the signals emitted upon binding of the two triphosphate pyrimidine nucleotides to the regulatory sites do not follow the same pathway and involve different mechanisms.
England
P
P
Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, C.N.R.S. URA 1682, Université Pierre et Marie Curie, Paris, France.
Hervé
G
G
D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors*; D003570:Cytidine Triphosphate / Q000008:administration & dosage*; D004357:Drug Synergism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D013329:Structure-Activity Relationship; D014544:Uridine Triphosphate / Q000008:administration & dosage* / Q000737:chemistry
D003570:Cytidine Triphosphate; D001221:Aspartate Carbamoyltransferase; D014544:Uridine Triphosphate