Controlled proteolysis of the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells: evidence for discrete structural domains.
The multifunctional protein that initiates de novo pyrimidine biosynthesis in mammalian cells carries carbamoylphosphate synthetase, aspartate transcarbamylase (aspartate carbamoyltransferase), and dihydro-orotase activities on a single 215,000-dalton polypeptide chain. Kinetic studies of the controlled proteolysis of the molecule by elastase showed that the protein was not attacked at random by the protease but rather was successively cleaved into at least six well-defined proteolytic fragments. The initial cleavage converted the intact molecule into a 190,000-dalton species which appeared to retain all of the catalytic and regulatory functions of the native protein. This species was subsequently cleaved into two fragments, 150,000 and 40,000 daltons. The 40,000-dalton species, which carried the aspartate transcarbamylase activity, was resistant to further proteolysis; the 150,000-dalton polypeptide, which carried carbamoyl-phosphate synthetase and dihydro-orotase activities, underwent further digestion to 140,000 daltons. Continued proteolysis produced two species, 79,000 and 45,000 daltons; like the 40,000-dalton species, these were stable against further elastase digestion. The aspartate transcarbamylase and dihydro-orotase activities and the regulatory functions were preserved throughout the course of digestion; the carbamoylphosphate synthetase activity was more labile. By using sucrose gradient centrifugation and ion exchange chromatography, the 40,000- and 45,000-dalton species have been isolated. The 40,000-dalton fragment was found to have only aspartate transcarbamylase activity; the 45,000-dalton fragment has only dihydro-orotase activity. These experiments showed that this multifunctional protein is organized as discrete structural domains in which regions of the polypeptide chain are autonomously folded into separate functional units.
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D002460:Cell Line; D002472:Cell Transformation, Viral; D006224:Cricetinae; D004080:Dihydroorotase / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism*; D008647:Mesocricetus; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011743:Pyrimidines / Q000096:biosynthesis*; D013539:Simian virus 40 / Q000235:genetics
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011743:Pyrimidines; D001221:Aspartate Carbamoyltransferase; D010196:Pancreatic Elastase; D000581:Amidohydrolases; D004080:Dihydroorotase; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia)