2DRC

INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS

2DRC の概要

• エントリーDOI: 10.2210/pdb2drc/pdb
• 分子名称: DIHYDROFOLATE REDUCTASE, CHLORIDE ION, METHOTREXATE, ... (5 entities in total)
• 機能のキーワード: oxidoreductase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 36982.44

構造登録者

Brown, K.A.,Kraut, J. (登録日: 1992-06-10, 公開日: 1994-01-31, 最終更新日: 2024-02-14)

主引用文献

Warren, M.S.,Brown, K.A.,Farnum, M.F.,Howell, E.E.,Kraut, J.
Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis.
Biochemistry, 30:11092-11103, 1991

PubMed Abstract: We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H). The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution. The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain. A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22. Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining. Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range. For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values. While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered. Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme. We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate.

PubMed: 1932031
DOI: 10.1021/bi00110a011

実験手法

X-RAY DIFFRACTION (1.9 Å)