NAG (N-acetyl-L-glutamate), the essential allosteric activator of the first urea cycle enzyme, CPSI (carbamoyl phosphate synthetase I), is a key regulator of this crucial cycle for ammonia detoxification in animals (including humans). Automated cavity searching and flexible docking have allowed identification of the NAG site in the crystal structure of human CPSI C-terminal domain. The site, a pocket lined by invariant residues and located between the central β-sheet and two α-helices, opens at the β-sheet C-edge and is roofed by a three-residue lid. It can tightly accommodate one extended NAG molecule having the δ-COO− at the pocket entry, the α-COO− and acetamido groups tightly hydrogen bonded to the pocket, and the terminal methyl of the acetamido substituent surrounded by hydrophobic residues. This binding mode is supported by the observation of reduced NAG affinity upon mutation of NAG-interacting residues of CPSI (recombinantly expressed using baculovirus/insect cells); by the fine-mapping of the N-chloroacetyl-L-glutamate photoaffinity labelling site of CPSI; and by previously established structure–activity relationships for NAG analogues. The location of the NAG site is identical to that of the weak bacterial CPS activator IMP (inosine monophosphate) in Escherichia coli CPS, indicating a common origin for these sites and excluding any relatedness to the binding site of the other bacterial CPS activator, ornithine. Our findings open the way to the identification of CPSI deficiency patients carrying NAG site mutations, and to the possibility of tailoring the activator to fit a given NAG site mutation, as exemplified here with N-acetyl-L(±)-β-phenylglutamate for the W1410K CPSI mutation.
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December 2009
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Research Article|
November 11 2009
Structural insight on the control of urea synthesis: identification of the binding site for N-acetyl-L-glutamate, the essential allosteric activator of mitochondrial carbamoyl phosphate synthetase1 Available to Purchase
Satu Pekkala;
Satu Pekkala
*Centro de Investigación Príncipe Felipe (CIPF), Avda. Autopista del Saler 16, Valencia 46012, Spain
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Ana I. Martínez;
Ana I. Martínez
*Centro de Investigación Príncipe Felipe (CIPF), Avda. Autopista del Saler 16, Valencia 46012, Spain
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Belén Barcelona;
Belén Barcelona
*Centro de Investigación Príncipe Felipe (CIPF), Avda. Autopista del Saler 16, Valencia 46012, Spain
†Centro de Investigación Biomédica en Red para Enfermedades Raras (CIBERER-ISCIII), C/Álvaro de Bazán, 10 Bajo, 46010 Valencia, Spain
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José Gallego;
José Gallego
*Centro de Investigación Príncipe Felipe (CIPF), Avda. Autopista del Saler 16, Valencia 46012, Spain
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Elena Bendala;
Elena Bendala
‡Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, Valencia 46010, Spain
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Igor Yefimenko;
Igor Yefimenko
*Centro de Investigación Príncipe Felipe (CIPF), Avda. Autopista del Saler 16, Valencia 46012, Spain
†Centro de Investigación Biomédica en Red para Enfermedades Raras (CIBERER-ISCIII), C/Álvaro de Bazán, 10 Bajo, 46010 Valencia, Spain
‡Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, Valencia 46010, Spain
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Vicente Rubio;
†Centro de Investigación Biomédica en Red para Enfermedades Raras (CIBERER-ISCIII), C/Álvaro de Bazán, 10 Bajo, 46010 Valencia, Spain
‡Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, Valencia 46010, Spain
3Correspondence may be addressed to either Javier Cervera (email [email protected]) or Vicente Rubio (email [email protected]).
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Javier Cervera
*Centro de Investigación Príncipe Felipe (CIPF), Avda. Autopista del Saler 16, Valencia 46012, Spain
†Centro de Investigación Biomédica en Red para Enfermedades Raras (CIBERER-ISCIII), C/Álvaro de Bazán, 10 Bajo, 46010 Valencia, Spain
3Correspondence may be addressed to either Javier Cervera (email [email protected]) or Vicente Rubio (email [email protected]).
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Publisher: Portland Press Ltd
Received:
June 17 2009
Revision Received:
September 11 2009
Accepted:
September 15 2009
Accepted Manuscript online:
September 15 2009
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 424 (2): 211–220.
Article history
Received:
June 17 2009
Revision Received:
September 11 2009
Accepted:
September 15 2009
Accepted Manuscript online:
September 15 2009
Citation
Satu Pekkala, Ana I. Martínez, Belén Barcelona, José Gallego, Elena Bendala, Igor Yefimenko, Vicente Rubio, Javier Cervera; Structural insight on the control of urea synthesis: identification of the binding site for N-acetyl-L-glutamate, the essential allosteric activator of mitochondrial carbamoyl phosphate synthetase. Biochem J 1 December 2009; 424 (2): 211–220. doi: https://doi.org/10.1042/BJ20090888
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