Cloning, expression, and functional interactions of the amidotransferase domain of mammalian CAD carbamyl phosphate synthetase.
The trpG-type amidotransferases, a homologous but structurally diverse family of molecules, catalyze glutamine hydrolysis to supply ammonia for many biosynthetic reactions. The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of a 243-kDa polypeptide that initiates de novo pyrimidine biosynthesis, has been cloned and expressed in Escherichia coli. Complementation studies showed that a functional protein was produced in vivo which could provide ammonia for carbamyl phosphate synthesis by the host CPSase synthetase subunit. The recombinant 38-kDa protein was identified by immunoblotting, but when purified to homogeneity, had marginal glutaminase activity. Titration of the E. coli CPSase synthetase subunit with the mammalian GLNase domain resulted in the formation of a fully active 1:1 stoichiometric stable complex which catalyzed the glutamine-dependent overall reaction. The hybrid, isolated by gel filtration, had kinetic parameters (KGLNm = 102 microM, KATPm = 1.8 mM, kcat = 5.7 s-1) similar to those of the native E. coli CPSase. Thus, the amidotransferase activity of mammalian CPSase is carried by an autonomous domain which folds independently. However, optimal catalytic activity requires association of the glutaminase and synthetase domains. The conservation of this linkage in the mammalian E. coli hybrid suggests that the subunit interfaces must be nearly identical in the eukaryotic and prokaryotic proteins.
Guy
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Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
Evans
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D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004080:Dihydroorotase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D005816:Genetic Complementation Test; D005972:Glutaminase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008647:Mesocricetus; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D001221:Aspartate Carbamoyltransferase; D005972:Glutaminase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)