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.