N-acetylglutamate-independent activity of carbamyl phosphate synthetase (ammonia): Implications for the kinetic assay of acetylglutamate

In the presence of Mn 2+, carbamyl phosphate synthetase (ammonia) catalyzes considerable carbamyl phosphate synthesis in the absence of the allosteric activator, N-acetylglutamate. Under standard conditions, the acetylglutamate-independent activity of a purified carbamyl phosphate synthetase preparation was 8 to 10% of the V max observed at saturating (1 m m) acetylglutamate. The product formed in the reaction was identified unequivocally as carbamyl phosphate. Standard conditions included 5 m m ATPMn and 1.5 m m excess Mn 2+. The highest rate of acetylglutamate-independent activity was observed at [excess Mn 2+] of 1.5 m m; increasing the [ATPMn] from 5 to 20 m m doubled the acetylglutamate-independent activity, to 18% of V max. Only 1 20 as much acetylglutamate-independent activity was observed when Mg 2+ was substituted for Mn 2+. When both Mn 2+ and Mg 2+ were present, the acetylglutamate-independent activity was less than when Mn 2+ alone was present. Measurement of acetylglutamate-dependent activity of carbamyl phosphate synthetase (ammonia) revealed that one-half V max with Mn 2+ was achieved at 17 μ m acetylglutamate (about one-fifth of the value reported with Mg 2+), and the V max with Mn 2+ under standard conditions was only 60% of that observed with Mg 2+. The high affinity of carbamyl phosphate synthetase for acetylglutamate in the presence of Mn 2+ has been used in the development of a sensitive, accurate method for the measurement of acetylglutamate in small quantities of mitochondrial extracts. This method is described in detail.