Controlled proteolysis of ammonia-dependent carbamoyl-phosphate synthetase I from Syrian hamster liver.
Ammonia-dependent carbamoyl-phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16; formerly EC 2.7.2.5) isolated from hamster liver mitochondria is comprised of identical 160 kDa polypeptide chains. Controlled proteolysis by elastase sequentially cleaved this molecule into a small number of specific fragments. The first cleavage led to a complete loss of enzymatic activity and the formation of a 145 kDa species that was subsequently degraded into 83 kDa and 62 kDa fragments. Very different results were obtained when proteolysis was carried out in the presence of saturating ATP, MgCl2, NH4Cl, and the activator N-acetyl-L-glutamate. These ligands stabilized the molecule 8-fold against elastase digestion. Moreover, only small amounts of the 145 kDa species were generated. Instead, the molecule was initially cleaved into a fully active 120 kDa species and a 40 kDa proteolytic fragment. The same species were found in limit digests conducted in the presence and absence of ligands, indicating that only the sequence of elastase cleavages differed. Comparison of digests conducted in the presence of each ligand alone and in combination, showed that while NH4Cl and N-acetyl-L-glutamate were necessary for maximal stabilization of the molecule, the altered digestion pattern was produced specifically by MgATP. The MgATP-induced change in digestion pattern correlated well with the steady-state ATP saturation curve, suggesting that the production of the 120 kDa species resulted from ATP binding to the active site. The effect of MgATP on the proteolysis of carbamoyl-phosphate synthetase I was not the result of an alteration in oligomeric structure, but the protection of two elastase cleavage sites. The results were interpreted on the basis of the primary structure recently determined elsewhere.
Evans
D R
DR
Department of Biochemistry, Wayne State University School of Medicine, Detroit, MI 48201.
Balon
M A
MA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000494:pharmacology; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008647:Mesocricetus; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*
D005971:Glutamates; D010446:Peptide Fragments; D000643:Ammonium Chloride; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D010196:Pancreatic Elastase; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D008274:Magnesium; C016195:N-acetylglutamic acid