Pressure-induced dissociation of carbamoyl-phosphate synthetase domains. The catalytically active form is dimeric.
Carbamoyl-phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs. The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in carbamoyl phosphate synthesis. When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H. I., and Evans, D. R. (1996) J. Biol. Chem. 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze carbamoyl phosphate synthesis. The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of carbamoyl phosphate required a homodimer of CPS.A or CPS.B. To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell. Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity. Activity was recovered once the protein was returned to atmospheric pressure. If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity. In contrast, the bicarbonate-dependent ATPase and the carbamoyl phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation. These experiments confirmed the hypothesis that the synthesis of carbamoyl phosphate requires the concerted action of the two active sites within the homodimer.
Guy
H I
HI
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Schmidt
B
B
Hervé
G
G
Evans
D R
DR
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000378:metabolism; D008322:Mammals; D011312:Pressure; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D011994:Recombinant Proteins; D005973:Glutamine; D000255:Adenosine Triphosphate; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)