Mechanism of allosteric modulation of Escherichia coli carbamoyl phosphate synthetase probed by site‐directed mutagenesis of ornithine site residues

FEBS Letters

Rubio, Vicente¹  Rochera, Lourdes²  Fresquet, Vicente²  Cervera, Javier² 

[1] Instituto de Biomedicina de Valencia (CSIC), Jaime Roig 11, Valencia 46010, Spain;Instituto de Investigaciones Citológicas (FVIB), Amadeo de Saboya 4, Valencia 46010, Spain

关键词: Carbamoyl phosphate synthetase;    Ornithine;    Allosteric regulation;    Site-directed mutagenesis;    Pyrimidine biosynthesis;    Arginine biosynthesis;    CPS;    carbamoyl phosphate synthetase;    CP;    carbamoyl phosphate;   

DOI  :  10.1016/S0014-5793(02)02392-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown