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Pressure-induced dissociation of carbamoyl-phosphate synthetase domains. The catalytically active form is dimeric.

Authors :
Guy HI
Schmidt B
Hervé G
Evans DR
Source :
The Journal of biological chemistry [J Biol Chem] 1998 Jun 05; Vol. 273 (23), pp. 14172-8.
Publication Year :
1998

Abstract

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.

Details

Language :
English
ISSN :
0021-9258
Volume :
273
Issue :
23
Database :
MEDLINE
Journal :
The Journal of biological chemistry
Publication Type :
Academic Journal
Accession number :
9603918
Full Text :
https://doi.org/10.1074/jbc.273.23.14172