Mammalian dihydroorotase; secondary structure, and interactions with other proteolytic fragments from the multienzyme polypeptide CAD.
We have purified mammalian dihydroorotase as a polypeptide fragment of 46 kDa from an elastase digest of CAD, the 240-kDa multienzyme that catalyses the first three reactions of pyrimidine biosynthesis. The thermal unfolding of the domain was analysed through the change in circular dichroism, indicating a sharp transition at 45 degrees C in which most of the native alpha-helix is lost. Although there is good evidence that the fragments associate as dimers in solution, chemical cross-linking was only possible when the dihydroorotase domain was included in a larger proteolytic fragment of 190-195 kDa. Cross-linking of the isolated domain yielded a species that appeared to result from links between two or more sub-domains, and did not yield the expected 90-kDa dimer of dihydroorotase. We speculate that the presence of other folded regions of CAD stabilises the interactions between dihydroorotase domains.
Hemmens
B
B
Department of Biochemistry, University of Dundee, Scotland.
Carrey
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry*; D002478:Cells, Cultured; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004080:Dihydroorotase / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D017433:Protein Structure, Secondary
C033626:CAD trifunctional enzyme; D003432:Cross-Linking Reagents; D009097:Multienzyme Complexes; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)