Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have prepared a selectively deuterated dihydrofolate reductase in which all the aromatic protons except the C(2) protons of tryptophan have been replaced by deuterium and have examined the 1H NMR spectra of its complexes with folate, trimethoprim, methotrexate, NADP+, and NADPH. One of the four Trp C(2)-proton resonance signals (signal P at 3.66 ppm from dioxane) has been assigned to Trp-21 by examining the NMR spectrum of a selectively deuterated N-bromosuccinimide-modified dihydrofolate reductase. This signal is not perturbed by NADPH, indicating that the coenzyme is not binding close to the 2 position of Trp-21. This contrasts markedly with the 19F shift (2.7 ppm) observed for the 19F signal of Trp-21 in the NADPH complex with the 6-fluorotryptophan-labeled enzyme. In fact the crystal structure of the enzyme . methotrexate . NADPH shows that the carboxamide group of the reduced nicotinamide ring is near to the 6 position of Trp-21 but remote from its 2 position. The nonadditivity of the 1H chemical-shift contributions for signals tentatively assigned to Trp-5 and -133 indicates that these residues are influenced by ligand-induced conformational changes.
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PMID:Proton nuclear magnetic resonance studies of the effects of ligand binding on tryptophan residues of selectively deuterated dihydrofolate reductase from Lactobacillus casei. 677 Aug 92

Escherichia coli dihydrofolate reductase contains five tryptophan residues distributed throughout its structure. In order to examine the regions of the protein surrounding these tryptophan residues, we have incorporated 6-fluorotryptophan into the protein. To assign the five resonances observed in the 19F NMR spectrum, five site-directed mutants of the enzyme were made, each with one tryptophan replaced by a phenylalanine. The 19F NMR spectra of the apoprotein, two binary complexes (with NADPH or methotrexate), and one ternary complex (with NADPH and methotrexate) were obtained. The chemical shifts of two of the tryptophan resonances (at positions 22 and 74) are particularly sensitive to ligand binding, while the remaining three (at positions 30, 47, and 133) change, but by less. Since several of the tryptophans are distant from the binding site, these results suggest that 19F NMR can detect ligand-induced changes that are propagated throughout the structure. In the apoprotein, the resonances of the tryptophans at positions 22 and 30 are broadened. In the binary complex with NADPH, the resonances of tryptophans 30 and 74 are broadened while that of tryptophan 22 almost disappears. The line broadening of the tryptophan 22 resonance may reflect motion in that part of the protein, since it is near a region that is disordered in the crystal structure of the apoprotein and its NADP+ complex. In contrast, in the ternary complex this region has a defined structure, and all resonances are of equal intensity and line width. The 19F NMR spectra of the apoprotein and the three ligand complexes were also examined as a function of urea concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:19F NMR spectroscopy of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate reductase: equilibrium folding and ligand binding studies. 818 Jan 72

Fluorine NMR experiments with a protein containing fluorinated amino acid analogs can often be used to probe structure and dynamics of the protein as well as conformational changes produced by binding of small molecules. The relevance of NMR experiments with fluorine-containing materials to characteristics of the corresponding native (nonfluorinated) proteins depends upon the extent to which these characteristics are altered by the presence of fluorine. The present work uses molecular dynamics simulations to explore the effects of replacement of tryptophan by 6-fluorotryptophan in folate and methotrexate complexes of the enzyme dihydrofolate reductase (DHFR) (Escherichia coli). Simulations of the folate-native enzyme complex produce local correlation times and order parameters that are generally in good agreement with experimental values. Simulations of the corresponding fluorotryptophan-containing system indicate that the structure and dynamics of this complex are scarcely changed by the presence of fluorinated amino acids. Calculations with the pharmacologically important methotrexate-enzyme complex predict dynamical behavior of the protein similar to that of the folate complex for both the fluorinated and native enzyme. It thus appears that, on the time scale sampled by these computer simulations, substitution of 6-fluorotryptophan for tryptophan has little effect on either the structures or dynamics of DHFR in these complexes.
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PMID:Effects of fluorine substitution on the structure and dynamics of complexes of dihydrofolate reductase (Escherichia coli). 928 25