Carbamoyl-phosphate synthetase I. Kinetics of binding and dissociation of acetylglutamate and of activation and deactivation.
The dissociation of the cofactor, acetylglutamate, from the enzyme-cofactor complex formed by carbamoyl-phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO-3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s-1) is considerably slower than the rate of enzyme turnover (approximately equal to 6 s-1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5-2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme-cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.
Britton
H G
HG
Instituto de Investigaciones Citologicas de la Caja de Ahorros de Valencia, Spain.
Rubio
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002498:Centrifugation; D002850:Chromatography, Gel; D004789:Enzyme Activation; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D051381:Rats
D005971:Glutamates; D000255:Adenosine Triphosphate; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D008274:Magnesium; C016195:N-acetylglutamic acid; D011188:Potassium