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 proteolytic fragment (mammalian aspartate transcarbamoylase) from the C-terminus of the 240-kDa multienzyme polypeptide carrying the activities carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydroorotase (CAD) is similar to that of the catalytic subunits from Escherichia coli aspartate transcarbamoylase. We have measured the extent of unfolding of the mammalian aspartate transcarbamoylase in guanidinium chloride solutions, and have also demonstrated that the protein cross-reacts with antibodies raised against the E. coli enzyme. CAD is digested 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 chromatography. Similar proteolytic cleavage and trimming by elastase releases a family of aspartate transcarbamoylase fragments. Direct N-terminal sequencing of the aspartate transcarbamoylase fragments confirms predictions 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 columns in a different fraction from the completely unphosphorylated preparation, demonstrating the affinity of the domains for each other.
Biochemistry Department, University of Dundee, Scotland.
B
B
Hemmens
E A
EA
Carrey
D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002942:Circular Dichroism; D006224:Cricetinae; D003429:Cross Reactions; D004080:Dihydroorotase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D010196:Pancreatic Elastase; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D017510:Protein Folding; D013045:Species Specificity; D014357:Trypsin
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D001221:Aspartate Carbamoyltransferase; D010196:Pancreatic Elastase; D014357:Trypsin; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)