Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers.

@article{Lewis1995MethotrexateresistantVO,
  title={Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers.},
  author={William S. Lewis and Vivian Cody and Nikolai Galitsky and Joe R Luft and Walter Pangborn and Srinivas K. Chunduru and H Trent Spencer and James R. Appleman and Raymond L. Blakley},
  journal={The Journal of biological chemistry},
  year={1995},
  volume={270 10},
  pages={
          5057-64
        }
}
Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (kcat) at pH 7.65 (except for the arginine 22 variant), but Km for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these… CONTINUE READING

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