Nucleotide ligands protect the inter-domain regions of the multifunctional polypeptide CAD against limited proteolysis, and also stabilize the thermolabile part-reactions of the carbamoyl-phosphate synthase II domains within the CAD polypeptide.
Improved methodologies are described which allow the measurement of the part-reactions, with glutamine or ammonia as nitrogen donor, of mammalian carbamoyl-phosphate synthase II (EC 6.3.5.5) through the incorporation of [14C]bicarbonate into either carbamoyl phosphate or carbamoylaspartate. The enzyme is part of the multifunctional polypeptide (CAD) which also comprises the pyrimidine-biosynthetic enzymes aspartate transcarbamoylase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3). The conformational stability of the carbamoyl-phosphate synthase was investigated through the inactivation of the part-reactions which occurred during incubation at 37 degrees C. The domain involved in the removal of the amide N from glutamine was more thermolabile than the ammonia-dependent synthase moiety. The former activity was stabilized in the presence of sodium aspartate or MgATP, whereas the latter was stabilized by MgATP and MgUTP. Binding of MgUTP and MgATP to CAD restricted the initial proteolysis by trypsin and elastase of one or both regions linking the carbamoyl-phosphate synthase domain to the other major domains. A model is described to account for both aspects of nucleotide binding to CAD; these stabilizing effects may be important in the cell, where similar concentrations of nucleotides are found.
D000818:Animals; D001221:Aspartate Carbamoyltransferase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004080:Dihydroorotase; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D008025:Ligases / Q000378:metabolism*; D008956:Models, Chemical; D009097:Multienzyme Complexes; D009711:Nucleotides / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000494:pharmacology; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
C033626:CAD trifunctional enzyme; D008024:Ligands; D009097:Multienzyme Complexes; D009711:Nucleotides; D011506:Proteins; D001221:Aspartate Carbamoyltransferase; D010196:Pancreatic Elastase; D014357:Trypsin; D004080:Dihydroorotase; D008025:Ligases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)