PROTEOLYTIC CLEAVAGE OF THE MULTIENZYME POLYPEPTIDE CAD TO RELEASE THE MAMMALIAN ASPARTATE TRANSCARBAMOYLASE - BIOCHEMICAL-COMPARISON WITH THE HOMOLOGOUS ESCHERICHIA-COLI CATALYTIC SUBUNIT

We have demonstrated biochemically that the conformation of the proteo lytic fragment (mammalian aspartate transcarbamoylase) from the C-term inus of the 240-kDa multienzyme polypeptide carrying the activities ca rbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihyd roorotase (CAD) is similar to that of the catalytic subunits from Esch erichia coli aspartate transcarbamoylase. We have measured the extent of unfolding of the mammalian aspartate transcarbamoylase in guanidini um chloride solutions, and have also demonstrated that the protein cro ss-reacts with antibodies raised against the E. coli enzyme. CAD is di gested by low concentrations of trypsin in the presence of 0.2 mM UTP to release an active aspartate transcarbamoylase domain and a 195-kDa 'nicked CAD' molecule containing active carbamoyl phosphate synthetase . These two products are easily separated by ion-exchange chromatograp hy. Similar proteolytic cleavage and trimming by elastase releases a f amily of aspartate transcarbamoylase fragments. Direct N-terminal sequ encing of the aspartate transcarbamoylase fragments confirms predictio ns of the most accessible residues in the region linking the aspartate transcarbamoylase and dihydroorotase domains. Only the largest of the four fragments generated by elastase retains phosphorylation site 2. When this largest fragment is phosphorylated, the family of aspartate transcarbamoylase fragments is eluted together from ion-exchange colum ns in a different fraction from the completely unphosphorylated prepar ation, demonstrating the affinity of the domains for each other.