N-acetylglutamate-independent activity of carbamyl phosphate synthetase (ammonia): implications for the kinetic assay of acetylglutamate.
In the presence of Mn2+, 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 Vmax observed at saturating (1 mM) acetylglutamate. The product formed in the reaction was identified unequivocally as carbamyl phosphate. Standard conditions included 5 mM ATPMn and 1.5 mM excess Mn2+. The highest rate of acetylglutamate-independent activity was observed at [excess Mn2+] of 1.5 mM; increasing the [ATPMn] from 5 to 20 mM doubled the acetylglutamate-independent activity, to 18% of Vmax. Only 1/20 as much acetylglutamate-independent activity was observed when Mg2+ was substituted for Mn2+. When both Mn2+ and Mg2+ were present, the acetylglutamate-independent activity was less than when Mn2+ alone was present. Measurement of acetylglutamate-dependent activity of carbamyl phosphate synthetase (ammonia) revealed that one-half Vmax with Mn2+ was achieved at 17 microM acetylglutamate (about one-fifth of the value reported with Mg2+), and the Vmax with Mn2+ under standard conditions was only 60% of that observed with Mg2+. The high affinity of carbamyl phosphate synthetase for acetylglutamate in the presence of Mn2+ has been used in the development of sensitive, accurate method for the measurement of acetylglutamate in small quantities of mitochondrial extracts. This method is described in detail.
D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D004789:Enzyme Activation; D005971:Glutamates / Q000032:analysis* / Q000378:metabolism; D008025:Ligases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D012588:Scintillation Counting
D005971:Glutamates; D008345:Manganese; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D008274:Magnesium; C016195:N-acetylglutamic acid