The objective is to elucidate the control mechanisms involved in the regulation of de novo pyrimidine biosynthesis in mammalian cells. The first three enzymes of the pathway, carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase are carried by a single 230 Kd polypeptide chain. This large multifunctional protein, called CAD, also exhibits homotropic and heterotropic allosteric transitions. Contiguous lengths of the polypeptide chain are separately folded into globular domains having specific functions. To map the domain structure of CAD, the molecule will be cleaved by controlled proteolysis into active fragments which will be isolated by HPLC and affinity chromatography. Moreover a library of monoclonal antibodies directed against different regions of the CAD molecule will be developed for immunoaffinity chromatography and for use as structural probes. The functional sites on each fragment will be mapped by enzyme assays, binding studies and specific chemical modification. The size and juxtaposition of each domain within the polypeptide will be established by HPLC peptide mapping, partial amino acid sequencing and immunochemical methods. Most of the primary structure of CAD will be determined by nucleotide sequencing of a nearly full length cDNA molecule. The oligomeric structure of CAD and its domains, the intra and interdomain interactions and the arrangement of domains with the intact complex will be investigated by electron microscopy, chemical crosslinking and hydrodynamic methods. To identify explain the aggregate functional properties such as channelling and reduction in transient time resulting from the association of the component enzymes, steady state, pre-steady state and reconstitution studies are planned. Comparative studies of the amino acid sequence of CAD with proteins of related function but different structure, correlation of the domain structure of CAD with the fine structure of its gene, and structural comparison of CAD with mitochondrial carbamyl phosphate synthetase may give insight into the origin of CAD. This research should provide a much clearer understanding of multifunctional proteins, a recently discovered class of molecules which are likely to have an important regulatory role in mammalian cells. Moreover CAD is the major locus of control in de novo pyrimidine biosynthesis, a pathway essential for cellular proliferation, so that this work may have implications in understanding proliferative disorders.
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