1dls
METHOTREXATE-RESISTANT VARIANTS OF HUMAN DIHYDROFOLATE REDUCTASE WITH SUBSTITUTION OF LEUCINE 22: KINETICS, CRYSTALLOGRAPHY AND POTENTIAL AS SELECTABLE MARKERS
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OverviewOverview
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.
DiseaseDisease
Known disease associated with this structure: Anemia, megaloblastic, due to DHFR deficiency (1) OMIM:[126060]
About this StructureAbout this Structure
1DLS is a Single protein structure of sequence from Homo sapiens with NDP and MTX as ligands. The following page contains interesting information on the relation of 1DLS with [Dihydrofolate Reductase]. Active as Dihydrofolate reductase, with EC number 1.5.1.3 Full crystallographic information is available from OCA.
ReferenceReference
Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers., Lewis WS, Cody V, Galitsky N, Luft JR, Pangborn W, Chunduru SK, Spencer HT, Appleman JR, Blakley RL, J Biol Chem. 1995 Mar 10;270(10):5057-64. PMID:7890613
Page seeded by OCA on Mon Nov 12 16:33:44 2007