Required allosteric effector site for N-acetylglutamate on carbamoyl-phosphate synthetase I.
Carbamoyl-phosphate synthetase I (CPSase I) catalyzes the entry and rate-limiting step in the urea cycle, the pathway by which mammals detoxify ammonia. One facet of CPSase I regulation is a requirement for N-acetylglutamate (AGA), which induces an active enzyme conformation and does not participate directly in the chemical reaction. We have utilized labeling with carbodiimide-activated [14C]AGA to identify peptides 120-127, 234-237, 625-630, and 1351-1356 as potentially being near the binding site for AGA. Identification of peptide 1351-1356 confirms the previous demonstration (Rodriquez-Aparicio, L. B., Guadalajara, A. M., and Rubio, V.(1989) Biochemistry 28, 3070-3074) that the C-terminal region is involved in binding AGA. Identification of peptides 120-127 and 234-237 constitutes the first evidence that the N-terminal region of the synthetase is involved in ligand binding. Since peptides 631-638 and 1327-1348 have been identified near the ATP site of CPSase I (Potter, M. D., and Powers-Lee, S. G.(1992) J. Biol. Chem. 267, 2023-2031), the present finding of involvement of peptides 625-630 and 1351-1356 at an "allosteric" activator site was unexpected. The idea that portions of the AGA effector site might be derived from an ancestral glutamine substrate site via a gene duplication and diversification event was considered.
McCudden
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Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Powers-Lee
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SG
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D005022:Ethyldimethylaminopropyl Carbodiimide; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008099:Liver / Q000201:enzymology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D051381:Rats
D003432:Cross-Linking Reagents; D005971:Glutamates; D008024:Ligands; D010446:Peptide Fragments; C016195:N-acetylglutamic acid; D005022:Ethyldimethylaminopropyl Carbodiimide