title: The function of Glu338 in the catalytic triad of the carbamoyl phosphate synthetase amidotransferase domain.
abstract: The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate. The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit. The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage. The mammalian-E. coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad. The mutant mammalian GLN domains formed stable complexes with the E. coli CPS subunit, but the catalytic activity was severely impaired. While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced. This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: A
authors.initials: A
authors.lastname: Hewagama
authors.forename: H I
authors.initials: HI
authors.lastname: Guy
authors.forename: M
authors.initials: M
authors.lastname: Chaparian
authors.forename: D R
authors.initials: DR
authors.lastname: Evans
mesh_terms: D000641:Ammonia / Q000378:metabolism; D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry* / Q000235:genetics; D002478:Cells, Cultured; D006224:Cricetinae; D004926:Escherichia coli / Q000201:enzymology*; D005972:Glutaminase / Q000737:chemistry* / Q000235:genetics; D005973:Glutamine / Q000494:pharmacology; D007700:Kinetics; D008647:Mesocricetus; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D005973:Glutamine; D000641:Ammonia; D005972:Glutaminase; D002222:Carbamoyl-Phosphate Synthase (Ammonia)