Dihydrofolate Reductase

Dihydrofolate Reductase (DHFR), a key enzyme in thymidine synthesis, catalyzes the NADPH dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) and, at much lower rate, the conversion of folate to THF. The reaction product, THF, is an essential cofactor in the conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) by thymidylate synthetase.^1,2 Therefore, DHFR is a critical enzyme in DNA synthesis and has become a target for drug development and cancer therapy. The variations between DHFR from different sources has enabled the development of species selective DHFR inhibitors, such as trimethoprim (antibacterial and antifungal), pyrimethamine (antiprotozoal), and methotrexate, MTX, (antineoplastic, antipsoriatic, and anti-inflammatory).³

Human DHFR is an 186 amino acid protein with an apparent molecular weight of 25 kDa. It is 30% homologous to the E. coli protein and up to 70% homologous to vertebrate proteins.² The human DHFR gene, as well as other mammalian DHFR genes, overcomes the inhibitory effects of methotrexate by the mechanism of gene amplification or by amino acid mutagenesis.³

This product is supplied as a solution in 10 mM Tris-HCl, pH 8.0, with 1 mM EDTA, 0.5 mM DTT, 5 µM NADPH, protease inhibitors, and 50% glycerol.

Kinetic Properties:

Km5,6 0.16 mM NADPH 0.03 mM 7,8-dihydrofolate 8-methylpterin 0.13 mM

Ki7 Folate 2.6x10-5 mM Methotrexate 6.1-9x10-9

References:

1. Gready, J.E., Dihydrofolate reductase: binding of substrates and inhibitors and catalytic mechanism. Adv. Pharmacol. Chem., 17, 37-103 (1980).
2. Blakley, R.L., Eukaryotic dihydrofolate reductase. Adv. Enzymol. Relat. Areas Mol. Biol., 70, 23-101 (1995).
3. Schweitzer, B.I., et al., Dihydrofolate reductase as a therapeutic target, FASEB J., 4, 2441-2452 (1990).
4. Hillcoat, B.L., et al., Effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase. Anal. Biochem., 21, 178-189 (1967).
5. T.J. Susten, S.S. Blankenship, D.T. Freisheim, J.H. Delcamp, Purification and characterization of dihydrofolate reductase from methotrexate-resistant human lymphoblastoid cells, Biochemistry, 22,633-639 (1983).
6. Thibault, V.; Koen, M.J.; Gready, J.E.Enzymic properties of a new mechanism-based substrate for dihydrofolate reductase, Biochemistry, 28, 6042-6049 (1989).
7. Jarabak, J. Bachur, N.R., A soluble dihydrofolate reductase from human placenta: purification and properties, Arch. Biochem. Biophys., 142, 417-425 (1971).

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