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Title: Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange

Abstract

The kinetic mechanism of carbamoyl-phosphate synthetase II from Syrian hamster kidney cells has been determined at pH 7.2 and 37 degrees C. Initial velocity, product inhibition, and dead-end inhibition studies of both the biosynthetic and bicarbonate-dependent adenosinetriphosphatase (ATPase) reactions are consistent with a partially random sequential mechanism in which the ordered addition of MgATP, HCO/sub 3/-, and glutamine is followed by the ordered release of glutamate and Pi. Subsequently, the binding of a second MgATP is followed by the release of MgADP, which precedes the random release of carbamoyl phosphate and a second MgADP. Carbamoyl-phosphate synthetase II catalyzes beta gamma-bridge:beta-nonbridge positional oxygen exchange of (gamma-/sup 18/O)ATP in both the ATPase and biosynthetic reactions. Negligible exchange is observed in the strict absence of HCO3- (and glutamine or NH/sub 4/+). The ratio of moles of MgATP exchanged to moles of MgATP hydrolyzed (nu ex/nu cat) is 0.62 for the ATPase reaction, and it is 0.39 and 0.16 for the biosynthetic reaction in the presence of high levels of glutamine and NH/sub 4/+, respectively. The observed positional isotope exchange is suppressed but not eliminated at nearly saturating concentrations of either glutamine or NH/sub 4/+, suggesting that this residual exchange results from either themore » facile reversal of an E-MgADP-carboxyphosphate-Gln(NH/sub 4/+) complex or exchange within an E-MgADP-carbamoyl phosphate-MgADP complex, or both. In the /sup 31/P NMR spectra of the exchanged (gamma-/sup 18/O)ATP, the distribution patterns of /sup 16/O in the gamma-phosphorus resonances in all samples reflect an exchange mechanism in which a rotationally unhindered molecule of (/sup 18/O, /sup 16/O)Pi does not readily participate.« less

Authors:
; ;
Publication Date:
Research Org.:
Smith Kline and French Laboratories, Swedeland, PA
OSTI Identifier:
6024828
Resource Type:
Journal Article
Journal Name:
Biochemistry; (United States)
Additional Journal Information:
Journal Volume: 26:9
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; LIGASES; BIOCHEMICAL REACTION KINETICS; ADP; ATP; ATP-ASE; BIOLOGICAL PATHWAYS; GLUTAMINE; HAMSTERS; ISOTOPIC EXCHANGE; KIDNEYS; NMR SPECTRA; OXYGEN 16; OXYGEN 18; PHOSPHATES; PHOSPHORUS 31; PROTEINS; ACID ANHYDRASES; AMIDES; AMINO ACIDS; ANIMALS; BODY; CARBOXYLIC ACIDS; ENZYMES; EVEN-EVEN NUCLEI; HYDROLASES; ISOTOPES; KINETICS; LIGHT NUCLEI; MAMMALS; NUCLEI; NUCLEOTIDES; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; OXYGEN COMPOUNDS; OXYGEN ISOTOPES; PHOSPHOHYDROLASES; PHOSPHORUS COMPOUNDS; PHOSPHORUS ISOTOPES; REACTION KINETICS; RODENTS; SPECTRA; STABLE ISOTOPES; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Meek, T D, Karsten, W E, and DeBrosse, C W. Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange. United States: N. p., 1987. Web. doi:10.1021/bi00383a026.
Meek, T D, Karsten, W E, & DeBrosse, C W. Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange. United States. https://doi.org/10.1021/bi00383a026
Meek, T D, Karsten, W E, and DeBrosse, C W. Tue . "Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange". United States. https://doi.org/10.1021/bi00383a026.
@article{osti_6024828,
title = {Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange},
author = {Meek, T D and Karsten, W E and DeBrosse, C W},
abstractNote = {The kinetic mechanism of carbamoyl-phosphate synthetase II from Syrian hamster kidney cells has been determined at pH 7.2 and 37 degrees C. Initial velocity, product inhibition, and dead-end inhibition studies of both the biosynthetic and bicarbonate-dependent adenosinetriphosphatase (ATPase) reactions are consistent with a partially random sequential mechanism in which the ordered addition of MgATP, HCO/sub 3/-, and glutamine is followed by the ordered release of glutamate and Pi. Subsequently, the binding of a second MgATP is followed by the release of MgADP, which precedes the random release of carbamoyl phosphate and a second MgADP. Carbamoyl-phosphate synthetase II catalyzes beta gamma-bridge:beta-nonbridge positional oxygen exchange of (gamma-/sup 18/O)ATP in both the ATPase and biosynthetic reactions. Negligible exchange is observed in the strict absence of HCO3- (and glutamine or NH/sub 4/+). The ratio of moles of MgATP exchanged to moles of MgATP hydrolyzed (nu ex/nu cat) is 0.62 for the ATPase reaction, and it is 0.39 and 0.16 for the biosynthetic reaction in the presence of high levels of glutamine and NH/sub 4/+, respectively. The observed positional isotope exchange is suppressed but not eliminated at nearly saturating concentrations of either glutamine or NH/sub 4/+, suggesting that this residual exchange results from either the facile reversal of an E-MgADP-carboxyphosphate-Gln(NH/sub 4/+) complex or exchange within an E-MgADP-carbamoyl phosphate-MgADP complex, or both. In the /sup 31/P NMR spectra of the exchanged (gamma-/sup 18/O)ATP, the distribution patterns of /sup 16/O in the gamma-phosphorus resonances in all samples reflect an exchange mechanism in which a rotationally unhindered molecule of (/sup 18/O, /sup 16/O)Pi does not readily participate.},
doi = {10.1021/bi00383a026},
url = {https://www.osti.gov/biblio/6024828}, journal = {Biochemistry; (United States)},
number = ,
volume = 26:9,
place = {United States},
year = {1987},
month = {5}
}