We are interested in investigating the mechanistic enzymology and regulatory mechanisms for the interconversion of PRPP to AIR, the first five steps in the purine biosynthetic pathway. We recently isolated a new trifunctional protein from chicken liver which possesses GAR synthetase, GAR transformylase and AIR synthetase activities, the second, third and fifth activities in the pathway. We propose to study the mechanism of these synthetases and hope to define their domains within the polypeptide. We hope to examine the possibility that PRA aminotransferase, 1st enzyme, and FGAM synthetase, 4th enzyme, in this pathway are physically linked, perhaps through their glutamine binding domains. Antibodies prepared to the purified amidotransferase and the cDNA isolated from chicken liver cDNA library will be used as probes to determine whether these activities are part of multidomained protein. The stability of three of the intermediates generated in the enzymatic conversion PRPP to AIR will be examined as a potential explanation for the existence of multidomained protein which may exist in multifunctional enzyme complexes. The mechanistic enzymology of each of the five activities will be investigated, and based on these results design of mechanism based inhibitors will be undertaken. New potent inhibitors of purine biosynthesis may be of use as chemotherapeutic agents and as aids in the investigation of the regulatory mechanisms involved in this pathway.
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