Methotrexate-resistant variants of human dihydrofolate reductase. Effects of Phe31 substitutions

S.K Chunduru,V Cody,J.R Luft,W Pangborn,J.R Appleman,R.L Blakley

doi:10.1016/s0021-9258(17)36916-8

S.K Chunduru, V Cody + Show 4 more

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https://doi.org/10.1016/s0021-9258(17)36916-8

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Abstract

Substitution of glycine or alanine for phenylalanine 31 in human dihydrofolate reductase produces variants that are inhibited less by methotrexate (MTX) than the previously reported serine variant. The 100 times decrease in MTX affinity for the glycine variant is due to slower binding, and to inability of the initial complex to isomerize to a nondissociating conformer. A polar group at position 31 is unnecessary for resistance, but residues larger than serine confer no resistance. The glycine variant best fulfills criteria for gene therapy: low Km for H2folate, high kcat, and good stability. Although kcat is unaltered by these mutations, the rate of hydride transfer is greatly decreased. Presteady-state measurements have enabled a complete catalytic scheme to be constructed for the glycine variant that predicts observed steady-state behavior. The crystal structures of inhibitor complexes of the serine, alanine, and glycine mutants and of the wild-type enzyme show that the mutations cause little perturbation of the protein backbone, of side chains of residues at the active site, or of the bound inhibitor. A molecule of bound water occupies the space vacated by the phenyl group.

Highlights

From the $Department of Molecular Pharmacology, St
Substitution of glycine or alanine for phenylalanine When cell lines are exposed to high concentratioonfsMTX, it 31 in human dihydrofolate reductase produces variantsis frequently possible to isolate sublines that are MTX-resisthat are inhibitedless by methotrexate (MIX) than the tant
Kcat is unaltered by these mutations, the ratoef hydride transfer is greatly decreased.Presteady-statemeasurements haveenabledacompletecatalyticscheme to beconfor this variant hDHFR, thiserinecreased affinity of the apoenzyme forH,folate, itsK, is increased

Summary

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Human DHFRsthat have been reported have been thoroughly examined in regard to allof these characteristics. DHFR with substitutionof amino acids with small side chains for Phe, and have examined their fitnessforuseingene transfer studies as measured by the criteria mentioned. The best of them isthe Gly31variant, and we have investigated with this variant the effectosf the mutation on critical steps inthe catalytic pathway, and on the formation and dissociatoifotnhe. We have sought an explanation for these effects in the crystal structure of complexes of some of these variants. The abbreviations used are: DHFRd,ihydrofolate reductase; hDHFR, human dihydrofolate reductase; wt, wild-type; H,folate, 7,8-. Dihydrofolate; H,folate, (6S)-5,6,7,8-tetrahydrofolateM; ATS2, 5 mM 2-(N-morpholino)ethanesulfonicacid, 25 mM acetate, 50 mM Tris, 100 mM NaCl, and 0.02% sodium azide; MTX, methotrexate; CB3717, 5,8-

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