Mechanism of allosteric modulation of Escherichia coli carbamoyl phosphate synthetase probed by site-directed mutagenesis of ornithine site residues.
The role of residues of the ornithine activator site is probed by mutagenesis in Escherichia coli carbamoyl phosphate synthetase (CPS). Mutations E783A, E783L, E892A and E892L abolish ornithine binding, E783D and T1042V decrease 2-3 orders of magnitude and E892D decreased 10-fold apparent affinity for ornithine. None of the mutations inactivates CPS. E783 mutations hamper carbamate phosphorylation and increase K(+) and MgATP requirements, possibly by perturbing the K(+)-loop near the carbamate phosphorylation site. Mutation E892A activates the enzyme similarly to ornithine, possibly by altering the position of K891 at the opening of the tunnel that delivers the carbamate to its phosphorylation site. T1042V also influences modulation by IMP and UMP, supporting signal transmission from the nucleotide effector to the ornithine site mediated by a hydrogen bond network involving T1042. Ornithine activation of CPS may be mediated by K(+)-loop and tunnel gating changes.
Rochera
Lourdes
L
Instituto de Investigaciones Citológicas (FVIB), Amadeo de Saboya 4, Valencia 46010, Spain.
Fresquet
Vicente
V
Rubio
Vicente
V
Cervera
Javier
J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation / Q000502:physiology; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009952:Ornithine / Q000378:metabolism*; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D009952:Ornithine; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D011188:Potassium