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.
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
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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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