This proposal outlines a research effort centered on folate requiring enzymes that serve as a basis for investigating in a broader context, fundamental issues in enzymic catalysis and mechanism and in the intracellular organization of biosynthetic pathways. Dihydrofolate reductase will be used to address the relationship between dynamic fluctuations in the protein structure and catalytic function. The study on a number of select dihydrofolate reductase mutants will combine and analyze data from diverse methods: kinetic evaluation of the steps in the catalytic cycle, dynamic NMR measurements, molecular dynamics simulations, and single molecule spectroscopy. 5-Aminoimidazole-4- carboxamide ribonucleotide transformylase will be studied to elucidate the identity of the catalytic residues (active site or substrate) and their mode of action in promoting the formyl transfer reaction from the 10- formyltetrahydrofolate cofactor to the ribonucleotide. Despite the chemical similarity in the reaction catalyzed by this transformylase and that requiring glycinamide ribonucleotide as its substrate, the structure, amino acid sequence, and mode of catalysis of the two enzymes is remarkably different raising questions about the ,evolution of this protein family. Both transformylase enzymes function in de novo purine biosynthesis, postulated like other biosynthetic pathways to exist as a multi-enzyme complex but strong supporting evidence for such a proposal is scant. In an effort to determine whether the enzymes in this pathway are complexed or diffuse in the cytoplasm of the bacterial or mammalian cell, both in vitro and in vivo measurements will be used: the former incorporates isothermal calorimetry and surface plasmon resonance methods, and the latter confocal fluorescence microscopy. Computer models will be developed based on the determination of enzyme and metabolite levels in the cell to predict the flux of intermediates in the pathway.
| Chan, Chung Yu; Pedley, Anthony M; Kim, Doory et al. (2018) Microtubule-directed transport of purine metabolons drives their cytosolic transit to mitochondria. Proc Natl Acad Sci U S A 115:13009-13014 |
| Pedley, Anthony M; Karras, Georgios I; Zhang, Xin et al. (2018) Role of HSP90 in the Regulation of de Novo Purine Biosynthesis. Biochemistry 57:3217-3221 |
| Mangold, Colleen A; Yao, Pamela J; Du, Mei et al. (2018) Expression of the purine biosynthetic enzyme phosphoribosyl formylglycinamidine synthase in neurons. J Neurochem 144:723-735 |
| Pedley, Anthony M; Benkovic, Stephen J (2017) A New View into the Regulation of Purine Metabolism: The Purinosome. Trends Biochem Sci 42:141-154 |
| French, Jarrod B; Jones, Sara A; Deng, Huayun et al. (2016) Spatial colocalization and functional link of purinosomes with mitochondria. Science 351:733-7 |
| Fu, Rong; Sutcliffe, Diane; Zhao, Hong et al. (2015) Clinical severity in Lesch-Nyhan disease: the role of residual enzyme and compensatory pathways. Mol Genet Metab 114:55-61 |
| Chan, Chung Yu; Zhao, Hong; Pugh, Raymond J et al. (2015) Purinosome formation as a function of the cell cycle. Proc Natl Acad Sci U S A 112:1368-73 |
| Zhao, Hong; Chiaro, Christopher R; Zhang, Limin et al. (2015) Quantitative analysis of purine nucleotides indicates that purinosomes increase de novo purine biosynthesis. J Biol Chem 290:6705-13 |
| French, Jarrod B; Zhao, Hong; An, Songon et al. (2013) Hsp70/Hsp90 chaperone machinery is involved in the assembly of the purinosome. Proc Natl Acad Sci U S A 110:2528-33 |
| Zhao, Hong; French, Jarrod B; Fang, Ye et al. (2013) The purinosome, a multi-protein complex involved in the de novo biosynthesis of purines in humans. Chem Commun (Camb) 49:4444-52 |
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