Transformylase Enzymes Dihydrofolate Reductase

Pennsylvania State University-Univ Park, University Park PA

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Abstract

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.