Dihydrofolate Reductase

A Paradigm for Drug Design
  • Stephen J. Benkovic
  • Carston R. Wagner
Part of the New Horizons in Therapeutics book series (NHTH)


Dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase) catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F). This enzyme is necessary for maintaining intracellular pools of H4F and its derivatives, which are essential cofactors in the one-carbon transfer reactions utilized in the biosynthesis of purines, thymidylate, and several amino acids. It is also the target enzyme for antifolate drugs such as the antineoplastic drug methotrexate (MTX) and the antibacterial drug trimethoprim (Scheme 1). Because of its biological and pharmacological importance, dihydrofolate reductase (DHFR) has been the subject of intensive structural and kinetic studies (Blakley, 1985). The structures of the Escherichia coli and the Lactobacillus casei enzymes have been determined to 1.7 Å for several binary and ternary complexes containing MTX and/or NADP+ (Bolin et al., 1982; Filman et al., 1982; Matthews et al., 1985). The primary sequences of DHFR for eight bacterial and vertebrate sources are also available for comparison (Blakley, 1985). In addition, a complete kinetic scheme for wild-type E. coil DHFR has been derived from pre-steady-state and steady-state kinetics (Fierke et al., 1987).


Ternary Complex Dihydrofolate Reductase Dissociation Rate Constant Hydride Transfer Antifolate Drug 
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  1. Albery, W. J., and Knowles, J. R., 1976, Evolution of enzyme function and the development of catalytic efficiency, Biochemistry 15: 5631–5640.PubMedCrossRefGoogle Scholar
  2. Andrews, J., Fierke, C. A., Birdsall, B., Feeney, J., Roberts, G. C. K., and Benkovic, 1989, A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei Biochemistry 28: 5743–5750.CrossRefGoogle Scholar
  3. Benkovic, S. J., Fierke, C. A., and Naylor, A. M., 1988, Mechanism of oxygen activation by pteridine-dependent monooxygenases, Acc. Chem. Res. 21: 101–107.CrossRefGoogle Scholar
  4. Blakley, R. L., 1985, Dihydrofolate reductase, in: Folates and Pterins ( R. L. Blakley and S. J. Benkovic, eds.), pp. 191–253, Wiley, New York.Google Scholar
  5. Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., and Kraut, J., 1982, Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution, J. Biol. Chem. 257: 13650–13663.PubMedGoogle Scholar
  6. Chen, J-T., Taira, K., lb, C-P. D., and Benkovic, S. J., 1987, Probing the functional role of phenylalanine-31 of Escherichia coli dihydrofolate reductase by site-directed mutagenesis, Biochemistry 26: 4093–4100.PubMedCrossRefGoogle Scholar
  7. Fersht, A. R., 1987, The hydrogen bond in molecular recognition, Trends Biochem. Sci. 12: 301–304.CrossRefGoogle Scholar
  8. Fierke, C. A., and Benkovic, S. J., 1989, Probing the functional role of threonine-113 of Escherichia coli dihydrofolate reductase for its effect on turnover efficiency, catalysis and binding, Biochemistry 28: 478–486.PubMedCrossRefGoogle Scholar
  9. FershtFierke, C. A., Johnson, K. A., and Benkovic, S. J., 1987, Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli, Biochemistry 26: 381–391.Google Scholar
  10. Filman, D. J., Bolin, J. T., Matthews, D. A., and Kraut, J., 1982, Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 Å resolution. Environment of bound NADPH and implications for catalysis, J. Biol. Chem. 257: 13663–13672.PubMedGoogle Scholar
  11. Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., and Kraut, J., 1986, Functional role of aspartic acid-27 in dihydrofolate reductase revealed by mutagenesis, Science. 231: 1123–1128.PubMedCrossRefGoogle Scholar
  12. Lilius, E-M., Multanen, V-M., and Toivonen, V., 1979, Quantitative extraction and estimation of intracellular nicotinamide nucleotides of Escherichia coli, Anal. Biochem. 99: 22–27.PubMedCrossRefGoogle Scholar
  13. Matthews, D. A., Bolin, T. J., Burridge, J. M., Filman, D. J., Volz, K. W., Kaufman, B. T., Beddell, C. R., Champness, J. N., Stammers, D. K., and Kraut, J., 1985, Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim, J. Biol. Chem. 260: 381–391.PubMedGoogle Scholar
  14. Mayer, R. J., Chen, J-T., Taira, K., Fierke, C. A., and Benkovic, S. J., 1986, Importance of a hydrophobic residue in binding and catalysis by dihydrofolate reductase, Proc. Natl. Acad. Sci. USA 83: 7718–7720.PubMedCrossRefGoogle Scholar
  15. Morrison, J. R, and Stone, S. R., 1988, Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate, Biochemistry 27: 5499–5506.PubMedCrossRefGoogle Scholar
  16. Murphy, D. J., and Benkovic, S. J., 1989, Studies of hydrophobic interactions via mutants of E. coli dihydrofolate reductase: Separation of binding and catalysis, Biochemistry 28: 3025–3031.PubMedCrossRefGoogle Scholar
  17. Russell, A. J., and Fersht, A. R., 1987, Rational modification of enzyme catalysis by engineering surface charge, Nature 328: 496–500.PubMedCrossRefGoogle Scholar
  18. Taira, K., and Benkovic, S. J., 1988, Evaluation of the importance of hydrophobic interactions in drug binding to dihydrofolate reductase, J. Med. Chem. 31: 129–137.PubMedCrossRefGoogle Scholar
  19. Taira, K., Fierke, C. A., Chen, J. T., Johnson, K. A., and Benkovic, S. J., 1987, On interpreting the inhibition of catalysis dihydrosolute reductase, Tr. Biochem. Sci. 12: 275–278.CrossRefGoogle Scholar
  20. Wu, Y., andHouk, K. N., 1987, Theoretical transition structures for hydride transfer to methyleneiminium ion from methylamine and dihydropyridine. On the nonlinearity of hydride transfers, J. Am. Chem. Soc. 109: 2226–2227.CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • Stephen J. Benkovic
    • 1
  • Carston R. Wagner
    • 1
  1. 1.Department of ChemistryPennsylvania State UniversityUniversity ParkUSA

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