Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange.
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, HCO3-, 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-18O]ATP in both the ATPase and biosynthetic reactions. Negligible exchange is observed in the strict absence of HCO3- (and glutamine or NH4+). 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 NH4+, respectively. The observed positional isotope exchange is suppressed but not eliminated at nearly saturating concentrations of either glutamine or NH4+, suggesting that this residual exchange results from either the facile reversal of an E-MgADP-carboxyphosphate-Gln(NH4+) complex or exchange within an E-MgADP-carbamoyl phosphate-MgADP complex, or both. In the 31P NMR spectra of the exchanged [gamma-18O]ATP, the distribution patterns of 16O in the gamma-phosphorus resonances in all samples reflect an exchange mechanism in which a rotationally unhindered molecule of [18O3, 16O]Pi does not readily participate. These results suggest that the formation of carbamate from MgATP, HCO3-, and glutamine proceeds via a stepwise, not concerted mechanism, involving at least one kinetically competent covalent intermediate, such as carboxyphosphate.
Meek
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Karsten
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DeBrosse
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D000818:Animals; D001221:Aspartate Carbamoyltransferase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002460:Cell Line; D004080:Dihydroorotase; D007553:Isotope Labeling; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008433:Mathematics; D009097:Multienzyme Complexes; D010103:Oxygen Isotopes; D011506:Proteins / Q000378:metabolism*; D011865:Radioisotope Dilution Technique
C033626:CAD trifunctional enzyme; D002250:Carbon Radioisotopes; D009097:Multienzyme Complexes; D010103:Oxygen Isotopes; D011506:Proteins; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D008025:Ligases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)