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| == Biological Role of Dihydrofolate Reductase and Antifolates== | | == Function of Alpha Helix== |
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| | In order to test the function of the alpha helix within the domain a test was done in order to compare results of uptake within the domain with alpha helix and without the helix. An easy way to test this was a antibiotic assay. By isolating colonies strictly of gram negative bacteria with the alpha helix, NalPβ, and isolating colonies strictly of gram negative bacteria without the alpha helix, NalPβΔhelix, they could plate these separately. From there it was possible to compare susceptibility to antibiotics by placing the small circular tabs of antibiotics on the plate and measuring the difference in how effect the antibiotics was in penetrating the cell. The more penetration would show less growth inhibition or more sensitivity to the antibiotics. When the alpha helix was removed there was much more sensitivity to antibiotics showing that the removal of it leads to a more open pore. |
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| In nucleotide metabolism, Thymine is formed through the methylation of dUMP resulting in dTMP (thymidylate), which can undergo phosphorylation forming dTTP, deoxyribose thymine triphosphate, commonly utilized in DNA synthesis and replication. Thymidylate synthesis is performed by thymidylate synthase, obtaining the methyl for the reaction from N5N10-methylene-tetrahydrofolate. Thymidylate synthase involves the oxidation of N5N10-methylene-THF forming the dihydrofolate (DHF) product through the transfer and reduction of the methylene to the methyl group of thymidylate, dTMP. While this dTMP generation reaction is crucial in nucleotide metabolism, equally important is the regeneration of the THF cofactor from the DHF product.
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| Dihydrofolate reductase (DHFR) is responsible for the reduction reaction that regenerates THF from DHF using NADPH. The subsequent act of serine hydroxymethyltransferase yields the starting N5N10- methylene-THF. DHFR is a biologically important molecule in the synthesis of dTMP and cell replication, and the inhibition of this enzyme halts dTMP synthesis. DHFR in most species occurs as an enzyme monomeric and monofuctional in nature; however, DHFR and thymidylate synthase are present in the form of a bifunctional enzyme in rare cases.
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| The relevance of dTMP synthesis in cellular replication overall makes it an important enzyme in cellular development and proliferation. The most rapidly replicating cells are most quickly utilizing their dTMP supply and therefore rely on the enzymes involved in dTMP synthesis more than slower growing cells. This increased dependence on these enzymes is accompanied by an increase sensitivity to their inhibition. This concept makes the enzymes involved in thmidylate synthesis such as dihydrofolate reductase, prime targets for cancer therapy. Inhibition of DHFR would result in the most rapidly replicating cells, cancer in most cases, rendered incapable of reproducing and eventual susceptible to cellular death. Antifolates are classified as molecules involved in blocking folic acid activity, and are in fact used in the cancer treatment<ref>Voet, D., Voet, J. G., & Pratt, C. W. (2008). Fundamentals of biochemistry: life at the molecular level (3rd ed.). Hoboken, NJ: Wiley. </ref>.
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| == Mechanism of Action == | | == Mechanism of Action == |
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