title: Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals.
abstract: Carbamoyl phosphate (CP), the essential precursor of pyrimidines and arginine, is made in Escherichia coli by a single carbamoyl phosphate synthetase (CPS) consisting of 41.4 and 117.7 kDa subunits, which is feed-back inhibited by UMP and activated by IMP and ornithine. The large subunit catalyzes CP synthesis from ammonia in three steps, and binds the effectors in its 15 kDa C-terminal domain. Fifteen site-directed mutations were introduced in 13 residues of this domain to investigate the mechanism of allosteric modulation by UMP and IMP. Two mutations, K993A and V994A, decreased significantly or abolished enzyme activity, apparently by interfering with the step of carbamate synthesis, and one mutation, T974A, negatively affected ornithine activation. S948A, K954A, T974A, K993A and K993W/H995A abolished or greatly hampered IMP activation and UMP inhibition as well as the binding of both effectors, monitored using photoaffinity labeling and ultracentrifugation binding assays. V994A also decreased significantly IMP and UMP binding. L990A, V991A, H995A, G997A and G1008A had more modest effects or affected more the modulation by and the binding of one than of the other nucleotide. K993W, R1020A, R1021A and K1061A were without substantial effects. The results confirm the independence of the regulatory and catalytic centers, and also confirm functional predictions based on the X-ray structure of an IMP-CPS complex. They prove that the inhibitor UMP and the activator IMP bind in the same site, and exclude that the previously observed binding of ornithine and glutamine in this site were relevant for enzyme activation. K993 and V994 appear to be involved in the transmission of the regulatory signals triggered by UMP and IMP binding. These effectors possibly change the position of K993 and V994, and alter the intermolecular contacts mediated by the regulatory domain.
authors.affiliation: Instituto de Investigaciones Citológicas (FVIB), Amadeo de Saboya 4, Valencia, 46010, Spain.
authors.forename: V
authors.initials: V
authors.lastname: Fresquet
authors.forename: P
authors.initials: P
authors.lastname: Mora
authors.forename: L
authors.initials: L
authors.lastname: Rochera
authors.forename: S
authors.initials: S
authors.lastname: Ramón-Maiques
authors.forename: V
authors.initials: V
authors.lastname: Rubio
authors.forename: J
authors.initials: J
authors.lastname: Cervera
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site / Q000187:drug effects; D019943:Amino Acid Substitution / Q000235:genetics; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005816:Genetic Complementation Test; D007291:Inosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D009952:Ornithine / Q000378:metabolism / Q000494:pharmacology; D019920:Photoaffinity Labels / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013816:Thermodynamics; D014461:Ultracentrifugation; D014542:Uridine Monophosphate / Q000378:metabolism / Q000494:pharmacology
chemical_list: D019920:Photoaffinity Labels; D007291:Inosine Monophosphate; D014542:Uridine Monophosphate; D009952:Ornithine; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)