title: Site-directed substitution of Ser1406 of hamster CAD with glutamic acid alters allosteric regulation of carbamyl phosphate synthetase II.
abstract: Ser1406 of the allosteric region of the hamster CAD enzyme, carbamyl phosphate synthetase II (CPSase), is known to be phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Metabolic labeling experiments described here demonstrate that CAD is phosphorylated in somatic cells in culture. Phosphorylation is stimulated by treating cells with 8-bromo-cAMP, a PKA activator. The stimulation is essentially prevented by pretreatment with H-89, a PKA specific inhibitor. Substitution of Ser1406 with alanine results in an enzyme with kinetics and allosteric regulation indistinguishable from unsubstituted CAD. However, substitution to glutamic acid increases CPSase activity by reducing the apparent Km (ATP). The UTP concentration required to give 50% inhibition is increased rendering this altered enzyme significantly less sensitive to feedback inhibition, but allosteric activation by PRPP is unaffected. While these data do not prove that Ser1406 is phosphorylated in vivo, they do indicate that a specific alteration at this residue can affect allosteric regulation.
authors.affiliation: Department of Microbiology and Immunology, Albert B. Chandler Medical Center, University of Kentucky, Lexington 40536-0084, USA.
authors.forename: L C
authors.initials: LC
authors.lastname: Banerjei
authors.forename: J N
authors.initials: JN
authors.lastname: Davidson
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry*; D002478:Cells, Cultured; D006224:Cricetinae; D005816:Genetic Complementation Test; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D013329:Structure-Activity Relationship; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D010754:Phosphoribosyl Pyrophosphate; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D014544:Uridine Triphosphate