Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain

The first two steps of the de novo pyrimidine biosynthetic pathway in Sacch aromyces cerevisiae are catalyzed by a 240-kDa bifunctional protein encoded by the ura2 locus. Although the constituent enzymes, carbamoyl phosphate s ynthetase (CPSase) and aspartate transcarbamoylase (ATCase) function indepe ndently, there are interdomain interactions uniquely associated with the mu ltifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP . Moreover, the intermediate carbamoyl phosphate is channeled from the CPSa se domain where it is synthesized to the ATCase domain where it is used in the synthesis of carbamoyl aspartate. To better understand these processes, a recombinant plasmid was constructed that encoded a protein lacking the a midotransferase domain and the amino half of the CPSase domain, a 100-kDa c hain segment. The truncated complex consisted of the carboxyl half of the C PSase domain fused to the ATCase domain via the pDHO domain, an inactive di hydroorotase homologue that bridges the two functional domains in the nativ e molecule. Not only was the "half CPSase" catalytically active, but it was regulated by UTP to the same extent as the parent molecule. In contrast, t he ATCase domain was no longer sensitive to the nucleotide, suggesting that the two catalytic activities are controlled by distinct mechanisms. Most r emarkably, isotope dilution and transient time measurements showed that the truncated complex channels carbamoyl phosphate. The overall CPSase-ATCase reaction is much less sensitive than the parent molecule to the ATCase bisu bstrate analogue, N-phosphonacetyl-L-aspartate (PALA), providing evidence t hat the endogenously produced carbamoyl phosphate is sequestered and channe led to the ATCase active site.