In the past funding period three new enzymes and a new intermediate have been discovered in the purine biosynthetic pathway in E. coli. The applicant's laboratory has established that PurK catalyzes the conversion of AIR in an ATP and bicarbonate dependent reaction to the N(5)-carbamate of AIR (N(5)-CAIR). N(5)-CAIR has a half life of 30 sec at 370 C and pH 7.5. PurE then catalyzes an isomerization of this compound to CAIR. The Benkovic and Smith laboratories have discovered a novel, non-folate requiring GAR transformylase. These discoveries, the availability of gram quantities of all of the enzymes in the pathway, and the ability to use these enzymes as reagents to make substrates have set the stage for the goals in the next funding period. The applicant will use a variety of rapid kinetics methods (msec time scale) including rapid stopped-flow UV-vis spectroscopy, rapid chemical quench followed by analysis using HPLC, EPR, Mossbauer, or NMR spectroscopies to examine a number of mechanistic questions. The applicant will investigate: (1) The mechanism of the unusual PurE catalyzed rearrangement reaction; (2) The role of glutamine delivery of """"""""NH3"""""""" to its substrates in PRPP-amidotransferases and FGAR- aminotransferase reactions; (3) The role of the propeptide in B. subtilis and mammalian amidotransferases; and (4) The role of the non- heme iron in and the mechanism of an alpha-ketoglutarate dioxygenase (thymine hydroxylase) involved in pyrimidine metabolism. In addition to the mechanistic work, the applicant will continue to use genetics and kinetic methods to investigate the importance of channeling of chemically unstable intermediates in the purine pathway both in vitro and in vivo. The applicant will continue studies of the channeling of PRA between the first two enzymes in the pathway: PRPP aminotransferase and GAR synthetase. The applicant will also investigate the importance of channeling of the newly discovered N(5)-CAIR between PurE and PurK. She will use enzymes from both prokaryotic and eukaryotic systems to address these questions.
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