Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor.
Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO-3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is less than 20% of that of the acetylglutamate-activated enzyme whereas Vmax for the ATPase activity is greater than 40% of that with acetylglutamate. Pulse-chase experiments with H14CO-3 show formation of less "active CO2" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20-30 microM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 microM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time. From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATPA is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the gamma-phosphoryl group of ATPB appears to be facilitated.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D004789:Enzyme Activation; D005971:Glutamates / Q000494:pharmacology*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D051381:Rats; D013997:Time Factors
D005971:Glutamates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008025:Ligases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D008274:Magnesium; C016195:N-acetylglutamic acid; D011188:Potassium