Mitochondrial Carbamoyl Phosphate Synthetase Activity in the Absence of N‐Acetyl‐ l ‐glutamate

Rat liver carbamoyl‐phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. K m values for ATP, Mg 2+ and K + are greatly increased, the K m for HCO − 3 is not changed much, and the K m for NH + 4 is markedly reduced. V max for the synthetase reaction is < 20% of that of the acetylglutamate‐activated enzyme whereas V max for the ATPase activity is > 40% of that with acetylglutamate. Pulse‐chase experiments with H 14 CO − 3 show formation of less “active CO 2 ” (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 ( K d =20–30μM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATP B (ATP B provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATP A (ATP A yields P i ) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 μM acetylglutamate the lag is much longer, but at 10 μM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATP A and the ATP B 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 ATP A is associated with the conversion of T to R. R · AcGlu differs from R in that transfer to carbamate of the γ‐phosphoryl group of ATP B appears to be facilitated.