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)

Chicken liver dihydrofolate reductase is rapidly and stoichiometrically inactivated by a substituted 4,6-diaminodihydrotriazine containing a terminal benzenesulfonylfluoride (DTBSF). The substrate dihydrofolate largely prevents the enzyme inhibition by DTBSF, whereas NADPH had no effect, indicating that the inhibitor is bound at or near the folate site. Using radiolabeled inhibitor between 1.0 and 1.2 mol was incorporated/mol of enzyme (Mr = 21,651), following treatment with 8 M urea at 75 degrees C. Digestion of the maleylated, radiolabeled inhibitor-enzyme complex with trypsin and subsequent gel filtration on Sephadex G-50 SF yielded a single major peak of radioactivity. The covalently modified limited tryptic peptide was subsequently purified to homogeneity using high performance liquid chromatography. The radiolabeled tryptic peptide had the following sequence: Asn-Glu-Tyr (DTBS)-Lys-Tyr-Phe-Gln-Arg (residues 29-36). Automated Edman degradation of this peptide revealed that the radioactivity derived from the inhibitor was released at Step 3, identifying tyrosine-31 as the specific site of covalent attachment of the affinity label.
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PMID:Affinity labeling of chicken liver dihydrofolate reductase by a substituted 4,6-diaminodihydrotriazine bearing a terminal sulfonyl fluoride. 702 56

Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv') have been engineered using model sFv proteins based upon the 26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonal antibodies. As part of the 741F8 sFv construction process, the PCR-amplified 741F8 VH gene was modified in an effort to correct possible primer-induced errors. Genetic replacement of the N-terminal beta-strand sequence of 741F8 VH with that from the FR1 of anti-c-erbB-2 520C9 VH resulted in a dramatic improvement of sFv folding yields. Folding in urea-glutathione redox buffers produced active sFv' with a protected C-terminal sulfhydryl, presumably as the mixed disulfide with glutathione. Disulfide-bonded (sFv')2 homodimers were made by disulfide interchange or oxidation after reductive elimination of the blocking group. Both 26-10 (sFv')2 and 741F8 (sFv')2 existed as stable dimers that were well behaved in solution, whereas 741F8 sFv and sFv' exhibited considerable self-association. The 741F8 sFv binds to the extracellular domain (ECD) of the c-erbB-2 oncogene protein, which is often overexpressed in breast cancer and other adenocarcinomas. The recombinant ECD was prepared to facilitate the analysis of 741F8 binding site properties; the cloned ECD gene, modified to encode a C-terminal Ser-Gly-His6 peptide, was transfected into Chinese hamster ovary cells using a vector that also expressed dihydrofolate reductase to facilitate methotrexate amplification. Optimized cell lines expressed ECD-His6 at high levels in a cell bioreactor; after isolation by immobilized metal affinity chromatography, final ECD yields were as high as 47 mg/l. An animal tumor model complemented physicochemical studies of 741F8 species and indicated increased tumor localization of the targeted 741F8 (sFv')2 over other monovalent 741F8 species.
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PMID:Engineering disulfide-linked single-chain Fv dimers [(sFv')2] with improved solution and targeting properties: anti-digoxin 26-10 (sFv')2 and anti-c-erbB-2 741F8 (sFv')2 made by protein folding and bonded through C-terminal cysteinyl peptides. 747 92

The activation and inactivation of dihydrofolate reductase from chicken liver during denaturation in a wide concentration range of urea are compared with changes in intrinsic fluorescence. At 2 M urea the enzyme is activated 3.6-fold and is stable up to 12 h in the activated form. At 4 M urea, the enzyme activity increases about 5-fold initially but the activated enzyme loses activity rapidly to a level well below that of the native enzyme. The activated enzyme is stabilized in presence of either DHF or NADPH. The Kd and Km of the enzyme for the substrates at various urea concentrations were determined and compared. In the presence of 3 M urea, the values of Kd for DHF and NADPH increase 4-fold and 10-fold, respectively, whereas the corresponding Km values increase 25-fold and 3-fold. A large increase in Vmax is mainly responsible for the activation. The inactivation and unfolding in urea are both biphasic processes. For the fast phase, the rate constant of inactivation is 10-fold greater than that of unfolding in 4 M urea. The effect of (NH4)2SO4 on the activation and unfolding of the enzyme was also studied. The results suggest that the active site of the enzyme is more easily perturbed by denaturants; and the activated enzyme appears to have a more open and flexible conformation at the active site, which is favorable for the full expression of the catalytic power of the enzyme. A scheme for the sequential activation and inactivation of DHFR accompanying its unfolding by increasing concentrations of urea is proposed.
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PMID:Activation of chicken liver dihydrofolate reductase in concentrated urea solutions. 754 58

Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k approximately 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-flow circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that approximately 20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.
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PMID:Stopped-flow NMR spectroscopy: real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase. 756 25

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

The rate-limiting steps in the folding of dihydrofolate reductase from Escherichia coli have been shown to involve the conversion of a set of four intermediates to a corresponding set of native conformers via four parallel channels [Jennings et al. (1993) Biochemistry 32, 3783-3789]. Fluorescence and absorbance studies of the unfolding and refolding of the C85S/C152E double mutant at various final urea concentrations reveal two slow folding reactions, two fewer than observed in the wild-type protein. Refolding in the presence of substoichiometric levels of the inhibitor methotrexate shows that the two remaining slow reactions correspond to two parallel channels which lead to a pair of native conformers capable of binding the inhibitor. A combination of stopped-flow circular dichroism and cofactor binding studies confirms that the four parallel channels observed in the wild-type protein have collapsed into two channels in the mutant. Kinetic and equilibrium studies of the single cysteine mutants suggest that replacements of Cysteine-85 which perturb the hydrophobic core containing this side chain are responsible for the simplification of the kinetic mechanism. These results demonstrate that at least two of the parallel folding channels in dihydrofolate reductase arise when tertiary structure develops and are not dependent upon cis/trans isomerization at prolyl peptide bonds.
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PMID:Collapse of parallel folding channels in dihydrofolate reductase from Escherichia coli by site-directed mutagenesis. 825 92

The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane. SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins. The product of this gene, Sec13p, is a cytosolic protein peripherally associated with membranes. We developed a cell-free Sec13p-dependent vesicle formation reaction. Sec13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a Sec13p-dihydrofolate reductase fusion protein, respectively. These fractions were unable to support vesicle formation from the ER unless cytosol containing Sec13p was added. Cytosolic Sec13p fractionated by gel filtration as a large complex of about 700 kD. Fractions containing the Sec13p complex restored activity to the Sec13p- dependent vesicle formation reaction. Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of Sec13p, suggesting that another member of the complex becomes limiting when Sec13p is overproduced. Overproduced, monomeric Sec13p was inactive in the Sec13p-dependent vesicle formation assay.
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PMID:Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro. 843 27

To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in the flexible loop (117-131) was substituted to valine and leucine by site-directed mutagenesis. Despite the increased hydrophobicity of the side chains, the free energy changes of unfolding of the two mutants (G121V and G121L) determined by urea denaturation at 15 degrees C were decreased by 1.22 and 0.38 kcal/mol, respectively, compared with that of the wild-type. Thermal denaturation temperature, as monitored by differential scanning calorimetry, was decreased by 2.4 and 5.2 degrees C for G121V and G121L, respectively, accompanying the decrease in enthalpy change of denaturation. These findings indicate that the structure of DHFR is destabilized by the mutations, predominantly due to the large decrease in enthalpy change of denaturation relative to entropy change of denaturation. The steady-state kinetic parameter in the enzyme reaction, Km, was not influenced but kcat was greatly decreased by these mutations, resulting in 240- and 52-fold decreases in kcat/Km for G121V and G121L, respectively. The main effect of the mutations appeared to be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of hydrophobic interaction.
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PMID:Effects of point mutation in a flexible loop on the stability and enzymatic function of Escherichia coli dihydrofolate reductase. 845 78

We have designed a fusion gene encoding a chimeric mitochondrial precursor protein (avidin fusion protein) that consists of the mitochondrial presequence followed by mouse dihydrofolate reductase, a spacer segment, and streptavidin. The avidin fusion protein synthesized in vitro formed a tetramer at the avidin moiety on incubation with biotin during or after the translation reaction. The avidin fusion protein purified from the Escherichia coli overexpresser cells also formed the tetramer on dilution from 6M urea into buffer containing biotin. In in vitro import experiments with isolated yeast mitochondria, the tetramer of the avidin fusion protein became stuck across both mitochondrial membranes, with its N-terminal dihydrofolate reductase moiety in the matrix and its C-terminal avidin moiety exposed on the mitochondrial surface. Accumulation of the translocation intermediate of the fusion protein inhibited the import of a mitochondrial precursor protein, and allowed us to estimate the number of mitochondrial import sites.
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PMID:Avidin fusion protein as a tool to generate a stable translocation intermediate spanning the mitochondrial membranes. 857 89

It has been reported that the activation of dihydrofolate reductase (DHFR) from L1210 mouse leukaemia cells by KCl or thiol modifiers is accompanied by increased digestibility by proteinases [Duffy, Beckman, Peterson, Vitols and Huennekens (1987) J. Biol. Chem. 262, 7028-7033], suggesting a loosening up of the general compact structure of the enzyme. In the present study, the peptide fragments liberated from the chicken liver enzyme by digestion with trypsin in dilute solutions of urea or guanidine hydrochloride (GuHCl) have been separated by FPLC and sequenced. The sequences obtained are unique when compared with the known sequence of DHFR and thus allow the points of proteolytic cleavage identified for the urea- and GuHCl-activated enzyme to be at or near the active site. It was also indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene 6-sulfonate that conformational changes at the active site in dilute GuHCl parallel GuHCl activation. The above results indicate that the activation of DHFR in dilute denaturants is accompanied by a loosening up of its compact structure especially at or near the active site, suggesting that the flexibility at its active site is essential for the full expression of its catalytic activity.
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PMID:Activation of chicken liver dihydrofolate reductase by urea and guanidine hydrochloride is accompanied by conformational change at the active site. 867 Jan 38


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