CAD is a multifunctional protein that catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. The protein, which has glutamine dependent carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, consists of six copies of a 243 kDa polypeptide which is organized into separate structural domains having discrete functions. CAD is also the major locus of control of the de novo pathway and is allosterically regulated by UTP, an inhibitor, and by PRPP, an activator, and by cAMP dependent protein kinase mediated phosphorylation. CAD activity closely parallels the growth rate of normal and neoplastic tissues and, consequently, the constituent enzymes continue to be potential targets of chemotherapeutic agents. The goal of this research is to understand the structural organization and regulatory mechanisms of this protein. Four major functional domains comprised of 15 subdomains have been mapped by a combination of controlled proteolysis, DNA sequencing, and functional studies. The successful expression of the full length CAD polypeptide and many of the domains and subdomains in E. coli, now presents an opportunity to learn much more about the function and structure of the components of the CAD complex and their interactions. Moreover, several informative hybrid and chimeric molecules, consisting of CAD domains and the corresponding regions of proteins from other species, have been constructed. Investigation of these proteins by a combined approach involving protein chemistry, mutagenesis, and kinetic studies, should establish the function of the subdomains and the location of the catalytic and regulatory sites.. A major effort will be devoted to the crystallization of CAD, CAD domains and subdomains, and homologous bacterial enzymes for x-ray structure determination. The regulatory mechanisms and functional linkages between allosteric control and phosphorylation will be investigated by kinetic and binding studies of the intact protein and the isolated regulatory subdomain. Although the domains function autonomously, there is a network of interdomain interactions which modulate their function and plays an important role in relating their activities. The mechanisms of interdomain signaling which coordinate the partial reactions involved in the synthesis of carbamyl phosphate and the compartmentation of unstable intermediates will be worked out in steady state/presteady state kinetic and spectroscopic studies of the intact protein, reconstituted complexes, and hybrid molecules.
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