title: The isolation and characterization of the aspartate transcarbamylase domain of the multifunctional protein, CAD.
abstract: The aspartate transcarbamylase activity of mammalian cells is carried by a large multifunctional protein, CAD, that catalyzes the first three steps in the de novo pyrimidine biosynthetic pathway. Controlled proteolysis of CAD cleaved the polypeptide chain into several separate structural domains which carried the individual activities of the complex. The aspartate transcarbamylase activity was associated with a 40,000-dalton proteolytic fragment, which kinetic studies showed was released in one of the first proteolytic cleavages. The species was purified to homogeneity by chromatography on carboxymethyl-Sephadex. The isolated species, designated the aspartate transcarbamylase domain, had a molecular weight under denaturing conditions of 40,000 +/- 1,500, a pI = 9.4, and a sedimentation coefficient (S20,w) of 6.2. The sedimentation coefficient suggested that the isolated domain was an oligomer consisting of two or three identical copies of the 40,000-dalton proteolytic fragment. The aspartate saturation curve obtained at a saturating concentration of carbamyl phosphate gave Km = 2.1 X 10(-2)M and Vmax = 119.5 mumol/min/mg, corresponding to a turnover number of 4,780 min-1. Like the aspartate transcarbamylase activity of CAD, the activity was strongly inhibited by high concentrations of aspartate. The corresponding parameters from the carbamyl phosphate saturation curve were Km = 2.07 X 10(-5)M, Vmax = 52.5 mumol/min/mg, and a turnover number of 2,153 min-1. The similarity of these parameters to those obtained from a steady state kinetic study of CAD indicated that the tertiary structure of this region of the polypeptide chain was largely preserved in the isolated species. In the absence of stabilizing agents, the half-life of aspartate transcarbamylase activity of CAD was 60.2 h, while that of the isolated domain was 10.6 h. This result suggested that there were interactions with other regions of the molecule which stabilized the structure of the aspartate transcarbamylase domain in the intact complex.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D006224:Cricetinae; D004080:Dihydroorotase; D007700:Kinetics; D008647:Mesocricetus; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification
chemical_list: D000596:Amino Acids; C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D011506:Proteins; D001221:Aspartate Carbamoyltransferase; D010196:Pancreatic Elastase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)