1DLS

METHOTREXATE-RESISTANT VARIANTS OF HUMAN DIHYDROFOLATE REDUCTASE WITH SUBSTITUTION OF LEUCINE 22: KINETICS, CRYSTALLOGRAPHY AND POTENTIAL AS SELECTABLE MARKERS

1DLS の概要

• エントリーDOI: 10.2210/pdb1dls/pdb
• 分子名称: DIHYDROFOLATE REDUCTASE, NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, METHOTREXATE, ... (4 entities in total)
• 機能のキーワード: oxido-reductase, oxidoreductase
• 由来する生物種: Homo sapiens (human)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 22599.40

構造登録者

Cody, V.,Galitsky, N.,Luft, J.R.,Pangborn, W. (登録日: 1995-01-25, 公開日: 1995-04-20, 最終更新日: 2024-02-07)

主引用文献

Lewis, W.S.,Cody, V.,Galitsky, N.,Luft, J.R.,Pangborn, W.,Chunduru, S.K.,Spencer, H.T.,Appleman, J.R.,Blakley, R.L.
Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers.
J.Biol.Chem., 270:5057-5064, 1995

PubMed Abstract: 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 variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.

PubMed: 7890613
DOI: 10.1074/jbc.270.10.5057

実験手法

X-RAY DIFFRACTION (2.3 Å)