This proposal outlines a research effort centered on three important folate requiring enzymes: two involved in de novo purine biosynthesis, the human and avian bifunctional 5'-aminoimidazole-4 carboxamide ribonucleotide transformylase (AICARTfase) inosine monophosphate cyclohydrolase, the mono- (E.coli) and trifunctional (human) proteins possessing glycinamide ribonucleotide transformylase (GARTfase) activity, and the third, dihydrofolate reductase from E.coli and human sources. Experiments are designed to: 1) elaborate the minimal kinetic schemes through pretransient and steady state kinetics for formyl transfer by the two transformylase enzymes in order to provide a basis for: studies with mutants aimed at implicating a putative proton shuttle mechanism in the action of GARTfase, assessing the effect of neighboring domains on the kinetic properties of a GARTfase active site imbedded in the polyfunctional human form of the protein, and establishing a kinetic and mechanistic identity between the GARTfase and AICARTfase enzymes despite an absence of sequence homology; 2) Gain evidence for a link between the dynamic fluctuations in the protein structure of dihydrofolate reductase as monitored by 3D NMR and its catalytic activity; and 3) seek data for channeling in a putative de novo purine biosynthetic complex through the technique of confocal fluorescent microscopy using fluorescently tagged native and mutant forms of human GARTfase and AICARTfase. These folate requiring enzymes are important in one carbon unit transfer and have been the target of chemotherapy. Dihydrofolate reductase in particular serves as a paradigm for understanding the fundamentals of enzyme catalysis.
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| 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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