title: Trapping an activated conformation of mammalian carbamyl-phosphate synthetase.
abstract: The amidotransferase or glutaminase domain (GLN domain) of mammalian carbamyl-phosphate synthetase II (CPSase II) catalyzes glutamine hydrolysis and transfers ammonia to the synthetase domain (CPS domain), where carbamyl phosphate formation is catalyzed in three consecutive reactions. The GLN and CPS domains are part of a single polypeptide and are connected via a 29-amino acid chain segment (GC linker). In contrast, the two comparable domains of Escherichia coli CPSase are not fused, but are separate, noncovalently associated subunits. To establish the function of the GC linker in mammalian CPSase, it was deleted, and the two domains were directly fused. The deletion mutant not only catalyzed glutamine-dependent carbamyl phosphate synthesis, but was activated 10-fold relative to its wild-type counterpart. However, ammonia-dependent synthesis of carbamyl phosphate was abolished, indicating that ammonia no longer had access to the active site on the CPS domain. The mutant was still sensitive to inhibition by the allosteric effector UTP, but was no longer activated by the allosteric effector phosphoribosyl pyrophosphate, although evidence indicated that the latter could bind to the enzyme. The linker appears to serve as a spacer that allows the complex to cycle between two conformations, an open low activity form in which the ammonia site on the CPS domain is accessible and an activated conformation in which the ammonia generated in situ from glutamine is directly channeled to the CPS active site and access to exogenous ammonia is blocked.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: H I
authors.initials: HI
authors.lastname: Guy
authors.forename: D R
authors.initials: DR
authors.lastname: Evans
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000378:metabolism*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry / Q000378:metabolism*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004080:Dihydroorotase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D008647:Mesocricetus; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D010754:Phosphoribosyl Pyrophosphate; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D014544:Uridine Triphosphate