Substructure of the amidotransferase domain of mammalian carbamyl phosphate synthetase.
The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of the 243-kDa CAD polypeptide, consists of a carboxyl half that is homologous to all trpG-type amidotransferases and an amino half unique to the carbamyl phosphate synthetases. The two halves of the mammalian GLNase domain have been cloned separately, expressed in Escherichia coli, and purified. The 21-kDa carboxyl half, the catalytic subdomain, is extraordinarily active. The kcat is 347-fold higher and the KGlnm is 40-fold lower than the complete GLNase domain. Unlike the GLNase domain, the catalytic subdomain does not form a stable hybrid complex with the E. coli CPSase synthetase subunit. Nevertheless, titration of the synthetase subunit with the catalytic subdomain partially restores glutamine-dependent CPSase activity. The 19-kDa amino half, the interaction subdomain, binds tightly to the E. coli CPSase large subunit. Thus, the GLNase domain consists of two subdomains which can autonomously fold and function. The catalytic subdomain weakly interacts with the synthetase domain and has all of the residues necessary for catalysis. The interaction subdomain is required for complex formation and also attenuates the intrinsically high activity of the catalytic subdomain and, thus, may be a key element of the interdomain functional linkage.
Guy
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Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
Evans
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D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011994:Recombinant Proteins
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005973:Glutamine; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)