Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Degradation of 7,8-dihydrofolate (H2folate) in the presence of dihydrofolate reductase (DHFR) has been shown due not to an oxygenase activity of the reductase as previously reported but to dismutation of H2folate to folate and 5,6,7,8-tetrahydrofolate (H4folate). The reaction can be followed spectrophotometrically or by analysis of the reaction mixture by high-performance liquid chromatography (HPLC). The products have also been isolated and characterized. Oxygen uptake during the reaction is much less than stoichiometric with H2folate disappearance and is attributed to autoxidation of the H4folate formed. The dismutation activity is a property of highly purified Streptococcus faecium DHFR isoenzyme 2 (but not isoenzyme 1) and of Lactobacillus casei DHFR, but not of bovine liver DHFR. The activity is dependent on tightly bound NADP+ and/or NADPH. Removal of the nucleotide results in loss of dismutation activity, which is restored by adding NADP+ or NADPH. Maximum activity is obtained when approximately 1 mol equiv of nucleotide is added per mol of DHFR. It is proposed that in the dismutation reaction bound NADP(H) is alternately reduced and oxidized by incoming molecules of H2folate with release of folate and H4folate, respectively. The relatively slow rate of folate formation presumably limits the rate of the overall reaction. The equilibrium constant for the dismutation reaction is 19.4 +/- 7.4 at 22 degrees C and pH 7.0. Calculation of standard oxidation-reduction potentials at pH 7 gave values of -0.230 V for the H2folate/H4 folate pair and -0.268 V for the folate/H2folate pair. The mechanism by which NADP+ is retained by the enzyme from some sources during purification procedures is unclear.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dismutation of dihydrofolate by dihydrofolate reductase. 643 72

The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.
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PMID:Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex. 643 42

Mammalian cells and tissues were found to have two pathways for the biosynthesis of tetrahydrobiopterin (BH4): (i) the conversion of GTP to BH4 by a methotrexate-insensitive de novo pathway, and (ii) the conversion of sepiapterin to BH4 by a pterin salvage pathway dependent on dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity. In a Chinese hamster ovary cell mutant lacking dihydrofolate reductase (DUKX-B11), endogenous formation of BH4 proceeds normally but, unlike the parent cells, these cells or extracts of them do not convert sepiapterin or 7,8-dihydrobiopterin to BH4. KB cells, which do not contain detectable levels of GTP cyclohydrolase or BH4 but do contain dihydrofolate reductase, readily convert sepiapterin to BH4 and this conversion is completely prevented by methotrexate. In supernatant fractions of bovine adrenal medulla, the conversion of sepiapterin to BH4 is completely inhibited by methotrexate. Similarly, this conversion in rat brain in vivo is methotrexate-sensitive. Sepiapterin and 7,8-dihydrobiopterin apparently do not enter the de novo pathway of BH4 biosynthesis and may be derived from labile intermediates which have not yet been characterized.
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PMID:Biosynthesis of tetrahydrobiopterin by de novo and salvage pathways in adrenal medulla extracts, mammalian cell cultures, and rat brain in vivo. 657 16

We have determined whether the gene encoding dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) varies spontaneously in gene copy number in cells in vitro. Cells were stained under nonselective conditions with fluoresceinated methotrexate, which binds quantitatively to dihydrofolate reductase. Cells with the highest fluorescence were collected by a fluorescence-activated cell sorter and subsequently grown in the absence of methotrexate. At no time during the experiment were the cells placed under metabolic stress. After 10 successive rounds of growth and sorting, the derived population showed a 50-fold increase in fluorescence intensity, was highly resistant to methotrexate, and was amplified 40-fold in content of dihydrofolate reductase gene. We also found that cells already having amplified genes can undergo increases or decreases in their fluorescence and in gene copy number even more rapidly (at rates as high as 3 X 10(-2) amplification events per cell division) than do parental cells (ca. 10(-3) events per division). We therefore conclude that gene amplification can occur spontaneously in cells and that the rate of its occurrence varies with gene copy number.
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PMID:Rapid spontaneous dihydrofolate reductase gene amplification shown by fluorescence-activated cell sorting. 657 9

5-Formyltetrahydrofolate promotes the net dissociation of methotrexate bound to dihydrofolate reductase in the Ehrlich ascites tumor (L. H. Matherly et al., Cancer Res., 43: 2694-2699, 1983). Treatment of Ehrlich tumor cells with glucose or inhibitors of electron transfer stabilized the association of the antifolate with dihydrofolate reductase as reflected by a 2-fold increased fraction of dihydrofolate reductase-bound methotrexate and an abolition of the 5-formyltetrahydrofolate-induced dissociation of the inhibitor-enzyme complex. Glucose and azide were also found to increase the intracellular ratio of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized nicotinamide adenine dinucleotide phosphate (NADP+) in the tumor approximately 8- and 11-fold, respectively. However, other agents which enhanced the association between methotrexate and its target enzyme were less effective in increasing the intracellular level of NADPH relative to NADP+. Micromolar concentrations of NADPH promoted methotrexate binding to the purified Ehrlich tumor dihydrofolate reductase. Bound methotrexate could be dissociated from the purified enzyme by 5-methyltetrahydrofolate but less readily by 5-formyltetrahydrofolate and only in the presence of reduced levels of NADPH relative to NADP+. The tetraglutamate derivative of 5-methyltetrahydrofolate was even more effective than the underivatized compound in dissociating methotrexate from dihydrofolate reductase. These findings suggest a critical role for the cellular oxidation-reduction state in determining the affinity of dihydrofolate reductase for methotrexate and thus the cellular sensitivity to the antifolate. In addition, the data are consistent with the possibility that dihydrofolate reductase is a key locus for intracellular competitive interactions between reduced folates and methotrexate during leucovorin rescue from the pharmacological effects of the antifolate.
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PMID:Role of the cellular oxidation-reduction state in methotrexate binding to dihydrofolate reductase and dissociation induced by reduced folates. 660 65

The synthesis of D-6-phospho-[1-14C]gluconate and the utilization of this compound in a novel assay procedure for dihydrofolate reductase is described. This new assay method couples reductase-dependent NADP+ production to the enzymatic and NADP+-dependent decarboxylation of D-6-phospho-[1-14C]gluconate. By several criteria it is demonstrated that [14C]CO2 release is directly proportional to dihydrofolate reductase activity. This coupled radiometric assay for dihydrofolate reductase is more sensitive than the commonly used spectrophotometric assay and offers a number of advantages over earlier radiometric methods.
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PMID:A coupled radiometric assay for dihydrofolate reductase based on the oxidative decarboxylation of D-6-phospho-[1-14C]gluconate. 663 3

The amino acid sequence of the NADP+-dependent enzyme ovine 6-phosphogluconate dehydrogenase has been determined by conventional direct protein sequence analysis of peptides resulting from digestion of the protein with trypsin and chemical cleavages with cyanogen bromide, hydroxylamine, and iodosobenzoic acid. The polypeptide contains 466 amino acids and its NH2 terminus is acetylated. The Candida utilis enzyme is inactivated by reaction of pyridoxal phosphate with two lysine residues (Minchiotti, L., Ronchi, S., and Rippa, M. (1981) Biochim. Biophys. Acta 657, 232-242). These residues are conserved in the ovine enzyme. In contrast to NAD+ dehydrogenases which have weakly related sequences and spatially related folds in their nucleotide-binding sites, no significant sequence homologies were detected between 6-phosphogluconate dehydrogenase and any of three other NADP+-requiring enzymes, glutamate dehydrogenase, p-hydroxybenzoate hydroxylase, and dihydrofolate reductase. This is in accord with structural data that show no spatial relationship between NADP+-binding sites in these enzymes.
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PMID:Amino acid sequence of ovine 6-phosphogluconate dehydrogenase. 668 25

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was partially purified from a cloned strain of pyrimethamine-sensitive Plasmodium chabaudi and a drug-resistant clone derived from it. A molecular weight of approximately 120000 was estimated by gel filtration for enzyme from both pyrimethamine-sensitive and resistant parasites. The specific activities of the crude enzyme at pH 7.4 were 2.7 +/- 0.8 and 1.4 +/- 0.6 nmol min-1 mg-1 protein for sensitive and resistant strains, respectively. Methotrexate titration (pH 7.4, 37 degrees C) indicated that the apparent turnover number of the enzyme from the sensitive parasites was 1229 +/- 322 mol min-1 mol-1 compared with 1238 +/- 179 mol min-1 mol-1 for the enzyme from the resistant parasites. There was therefore no significant difference in the amounts of the enzyme from both sources. The Km value for dihydrofolate (9.3 microM) of the enzyme from the drug-sensitive parasites at pH 7.4 was lower than that from the resistant parasites by a factor of approximately 4. The Km values for NADPH of the enzyme from both sources were similar. Inhibition by pyrimethamine of the enzyme from the sensitive parasites was competitive with dihydrofolate, with Ki of 0.26 nM. By contrast, noncompetitive inhibition was observed for the enzyme from the resistant parasites, with Kis of 50 nM and Kii of 33 nM. The enzyme from drug-sensitive and drug-resistant parasites had different activity profiles with respect to pH and temperature. Moreover, the former was more sensitive to heat denaturation than the latter. From these results, it was concluded that the major basis for drug resistance is not an increase in enzyme content, but a large decrease in drug binding with the structurally different enzyme.
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PMID:Kinetic and molecular properties of dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi. 672 25

The kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been investigated by using progress curve, initial velocity, product inhibition, and dead-end inhibition studies as well as isotope effects. The results indicate that the reaction conforms to a random mechanism involving two dead-end complexes, viz., enzyme-DHF-THF and enzyme-NADP-DHF. At higher concentrations, DHF causes substrate inhibition by combining at the NADPH binding site on the enzyme. The steady-state velocity data can be analyzed adequately on the basis that rapid-equilibrium conditions apply. However, this can be only an approximate description of the reaction since the isotope effects observed with NADPD demonstrate clearly that catalysis cannot be rate limiting at pH 7.4. The choice of conditions for analysis of progress-curve data is discussed in the Appendix.
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PMID:Kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli. 675 19

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


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