The first three enzymatic steps in the de novo synthesis of pyrimidines (carbamyl phosphate synthetase II, aspartate transcarbamylase, dihydroorotase) are the same in all cells, whether prokaryotic or eukaryotic. The structure, organization, and regulation of these three enzymatic activities and their respective genes vary among species. In bacteria, each enzyme activity is associated with a separate protein. In mammals, they are found together in a single translation product of 240,000 daltons called CAD. By placing portions of hamster CAD cDNA into expression vectors, one can attempt to complement E. coli and hamster cell mutants defective in each of the three enzyme activities. This avenue of experimentation allows one (i) identify the minimum genetic information encoding each domain, (ii) to learn pyrimidine metabolism is affected when specific mutations are introduced that eliminate allosteric regulation of CAD or alter activity of one of its enzymes and (iii) to gain insight into the roles of the bridging sequences that link the enzymatic domains. Combining the approaches of molecular biology, genetics, and biochemistry should provide insight about the structure, function and evolution of a multifunctional protein that plays a key role in an important biosynthetic pathway.
