Carbamyl phosphate synthetase A of Neurospora crassa.
Carbamyl phosphate synthetase A of Neurospora crassa was partially purified from mitochondrial extracts. It is an extremely unstable enzyme (t 1/2 = 45 min at 25 detrees C) made up of two unequal subunits. The native enzyme has a molecular weight of approximately 175,000, and the large subunit has a molecular weight of about 125,000. Both the native enzyme and its large subunit are quite asymmetric, as revealed by slow sedimentation in sucrose gradents (7.3S and 6.6S, respectively). The small subunit has not been identified physically as a separate entity. The denaturation of the native, glutamine-dependent activity is correlated with dissociation of subunits, the larger of which retains a more stable, ammonia-dependent activity. Neither substrates nor any other agents except glycerol or polyethylene glycol appreciably stabilized the glutamine-dependent activity. Kinetic studies showed the native enzyme to have a Km for glutamine of about 0.16 mM, and a Km for NH4Cl of about 16 mM, at the optimal pH, 8.0. The enzyme, using either N donor, has a K+ requirement for activity, for which NH4+ can substitute. The glutamine leads to glutamate reaction, which requires the small subunit, also requires the large subunit and all reaction substrates for optimal activity. Other evidences of subunit interaction are the greater activity of the native enzyme, as opposed to the large subunit, with low concentrations of adenosine 5'-triphosphate-Mg2+, and in the stimulation of the ammonia-dependent activity of the native enzyme by glycine. Curiously, although the enzyme's role in biosynthesis is confined to the arginine pathway, it is completely indifferent to arginine or its precursors as feedback effectors or activators. The enzyme is compared with carbamyl phosphate synthetases of other organisms.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000643:Ammonium Chloride / Q000378:metabolism; D001639:Bicarbonates / Q000494:pharmacology; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000302:isolation & purification / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010770:Phosphotransferases / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology
D001639:Bicarbonates; D000643:Ammonium Chloride; D005973:Glutamine; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D011188:Potassium