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)

To test the possibility that long-range interactions might influence the folding and stability of dihydrofolate reductase, a series of single and double mutations at positions 28 and 139 were constructed and their urea-induced unfolding reactions studied by absorbance and circular dichroism spectroscopy. The alpha carbons of the two side chains are separated by 15 A in the native conformation. The replacement of Leu 28 by Arg and of Glu 139 by Gln resulted in additive effects on both kinetic and equilibrium properties of the reversible unfolding transition; no evidence for interaction was obtained. In contrast, the Arg 28/Lys 139 double replacement changed the equilibrium folding model from two state to multistate and showed evidence for interaction in one of the two kinetic phases detected in both unfolding and refolding reactions. The results can be explained in terms of a long-range, repulsive electrostatic interaction between the cationic side chains at these two positions.
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PMID:Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase. 269 6

We have purified milligram amounts of an importable mitochondrial precursor protein [the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (DHFR)]. This has made it possible, for the first time, to perform detailed studies on the conformation of a precursor protein and its interaction with lipid membranes. The precursor protein closely resembled authentic mouse DHFR with respect to secondary structure (measured by CD spectra) and stability towards urea (measured by tryptophan fluorescence and enzyme activity). With this precursor protein, the presequence thus does not significantly alter the folding of the attached 'passenger protein'. In contrast to the corresponding presequence peptide, the native precursor exhibited only weak ability to disrupt vesicles with a low mol% of negatively charged lipids, suggesting that the passenger protein masks the amphiphilic properties of the presequence. The membrane-perturbing properties of the precursor were greatly enhanced by increasing the vesicles' content of negatively charged lipid or by denaturing the precursor in 5 M urea. Interaction with vesicles rich in acidic phospholipid was accompanied by partial unfolding of the precursor, suggesting that such a conformational change may also be involved in the interaction of the precursor with the mitochondrial membranes.
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PMID:Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding. 284 Nov 14

Dihydrofolate reductase (DHFR; EC 1.5.1.3) was purified to homogeneity from soybean seedlings by affinity chromatography on methotrexate-aminohexyl Sepharose, gel filtration on Ultrogel AcA-54, and Blue Sepharose chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme gave a single protein band corresponding to a molecular weight of 22,000. The enzyme is not a 140,000 Da heteropolymer as reported by others. Amino acid sequence-specific antibodies to intact human DHFR and also antibodies to CNBr-generated fragments of human DHFR bound to the plant enzyme on Western blots and cross-reacted significantly in immunoassays, indicating the presence of sequence homology between the two enzymes. The plant and human enzymes migrated similarly on nondenaturing polyacrylamide electrophoretic gels as monitored by activity staining with a tetrazolium dye. The specific activity of the plant enzyme was 15 units/mg protein, with a pH optimum of 7.4. Km values of the enzyme for dihydrofolate and NADPH were 17 and 30 microM, respectively. Unlike other eukaryotic enzymes, the plant enzyme showed no activation with organic mercurials and was inhibited by urea and KCl. The affinity of the enzyme for folate was relatively low (I50 = 130 microM) while methotrexate bound very tightly (KD less than 10(-10) M). Binding of pyrimethamine to the plant enzyme was weaker, while trimethoprim binding was stronger than to vertebrate DHFR. Trimetrexate, a very potent inhibitor of the human and bacterial enzymes showed weak binding to the plant enzyme. However, certain 2,4-diaminoquinazoline derivatives were very potent inhibitors of the plant DHFR. Thus, the plant DHFR, while showing similarity to the vertebrate and bacterial enzymes in terms of molecular weight and immunological cross-reactivity, can be distinguished from them by its kinetic properties and interaction with organic mercurials, urea, KCl and several antifolates.
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PMID:Purification and characterization of dihydrofolate reductase from soybean seedlings. 310 22

Substitution of cysteine for proline-39 in Escherichia coli dihydrofolate reductase by oligonucleotide-directed mutagenesis positions the new cysteine adjacent to already existing cysteine-85. When the mutant protein is expressed in the E. coli cytosol, the cysteine sulfur atoms are found, by X-ray crystallographic analysis, to be in van der Waals contact but not covalently bonded to one another. In vitro oxidation by dithionitrobenzoate results in formation of a disulfide bond between residues 39 and 85 with a geometry close to that of the commonly observed left-handed spiral. Comparison of 2.0-A-refined crystal structures of the oxidized (cross-linked) and reduced (un-cross-linked) forms of the mutant enzyme shows that the conformation of the enzyme molecule was not appreciably affected by formation of the disulfide bond but that details of the molecule's thermal motion were altered. The disulfide-cross-linked enzyme is at least 1.8 kcal/mol more stable with respect to unfolding, as measured by guanidine hydrochloride denaturation, than either the wild-type or the reduced (un-cross-linked) mutant enzyme. Nevertheless, the cross-linked form is not more resistant to thermal denaturation. Moreover, the appearance of intermediates in the guanidine hydrochloride denaturation profile and urea-gradient polyacrylamide gels indicates that the folding/unfolding pathway of the disulfide-cross-linked enzyme has changed significantly.
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PMID:An engineered disulfide bond in dihydrofolate reductase. 330 20

The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Folding of dihydrofolate reductase from Escherichia coli. 353 77

The various interactions of rat liver dihydrofolate reductase with two unconjugated 7,8-dihydropteridines, 7,8-dihydrobiopterin and 6-methyl-7,8-dihydropteridine, have been compared with those of 7,8-dihydrofolate and folate. Of particular interest was the reactivity demonstrated by 7,8-dihydrobiopterin because of the potential physiological significance of this reaction both in the regeneration of tetrahydrobiopterin, a cofactor for various biological hydroxylations, and as a step in the biosynthesis of this compound from GTP. Kinetic experiments gave Km values of 0.17, 6.42, and 10.2 microM for 7,8-dihydrofolate, 7,8-dihydrobiopterin, and 6-methyl-7,8-dihydropteridine, respectively, with Vmax = 6.22, 2.39, and 1.54 mumol min-1 mg-1. With folate the enzyme showed high affinity (Km = 0.88 microM) but low Vmax (0.20 mumol min-1 mg-1). The natural cofactor was NADPH and a Km of approximately 0.7 microM was measured with each substrate. The enzyme was activated by both p-hydroxymercuribenzoate and urea when assayed with 7,8-dihydrofolate but was inhibited when 7,8-dihydrobiopterin was the substrate. The pH optimum for dihydrofolate reduction was 4 with enhancement at pH greater than or equal to 5.5 in the presence of 1 M NaCl. Peak activity with 7,8-dihydrobiopterin occurred at pH 4.8; this was shifted to pH 5.3 but was not enhanced by 1 M NaCl. Inhibition with methotrexate was similar whether the enzyme was assayed with either the conjugated or unconjugated 7,8-dihydro derivatives. The rat liver enzyme, highly unstable after purification, was stabilized in the presence of the nonionic detergent, Tween-20 (0.1%); however, the comparative properties toward the conjugated and unconjugated substrates were not altered by this treatment.
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PMID:Comparative activity of rat liver dihydrofolate reductase with 7,8-dihydrofolate and other 7,8-dihydropteridines. 397 May 30

Dihydrofolate reductase (EC 1.5.1.3; 5,6,7,8-tetrahydrofolate:NADP(+) oxidoreductase) from antifolate-resistant Lactobacillus casei has been isolated in pure form and examined in solution by high resolution proton magnetic resonance spectroscopy. The 220 MHz proton magnetic resonance spectrum of this small enzyme (about 15,000 daltons) consists of several distinct resonance peaks that provide a sensitive nonperturbing probe of its conformational state. Comparison of catalytically active enzyme with preparations denatured in 6 M urea demonstrates dramatically the overall contribution of secondary and tertiary structure to its proton magnetic resonance spectra. More subtle differences existing among several catalytically active enzyme forms may also be readily differentiated by proton magnetic resonance spectroscopy, e.g., the spectra of the ligand-free enzyme and forms containing stoichiometric amounts of tightly bound folate and dihydrofolate, each obtained separately by affinity chromatography, are easily identified. Addition of ligands to these spectroscopically distinct forms may induce changes in their proton magnetic resonance spectra. For example, addition of equimolar dihydrofolate to the apoenzyme converts its relatively featureless aromatic proton magnetic resonance spectrum to one indistinguishable from that of the original enzyme-dihydrofolate binary complex obtained chromatographically. Interaction of the pyridine nucleotide coenzymes NADP(+) or NADPH or of the antifolate Methotrexate with apoenzyme induces additional distinct spectral changes. Enzyme-NADPH and enzyme-Methotrexate binary complexes, which have different aromatic region proton magnetic resonance spectra, are converted to ternary complexes having quite similar spectra by addition of Methotrexate and NADPH, respectively, thus suggesting that an ordered addition of ligands is not required.
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PMID:Conformational changes induced in dihydrofolate reductase by folates, pyridine nucleotide coenzymes, and methotrexate. 415 40

The concentration of immunoreactive protein in the cytosol of L1210 cells measured using a specific radioimmunoassay for dihydrofolate reductase was substantially greater than the concentration of active enzyme which was measured by the binding of [3H]methotrexate. When the cytosol was subjected to gel filtration, two immunoreactive proteins were separated, a high-molecular-weight (Mr 318,000) protein which did not have catalytic activity and which did not bind [3H]methotrexate and a smaller protein (Mr approximately 20,000) which did reduce [3H]folic acid to tetrahydrofolate and did bind [3H]methotrexate. The nonfunctional high-molecular-weight protein neutralized the inhibitory effect of the antiserum on active dihydrofolate reductase. There was no spontaneous disaggregation of the big species into smaller subunits nor did 8 M urea alone, dithioerythritol alone, boiling with a mixture of 8 M urea and dithioerythritol, or RNase alter its apparent molecular weight. Trypsin, however, digested both the nonfunctional and active immunoreactive forms of the enzyme. Isoelectric focusing of the cytosol separated two nonfunctional immunoreactive isoproteins, each having the same isoelectric points as the two active isoenzymes of dihydrofolate reductase (pls of 8.0 and 8.5). Studies in rapidly replicating and stationary-phase L1210 cells showed that the concentration of the nonfunctional immunoreactive protein increased rapidly, reaching a peak on Day 2 of log growth at which time active enzyme was at a nadir, and then decreased rapidly, reaching a nadir on Day 4, at which time active enzyme was at a peak. The identical isoelectric points for the inactive and active immunoreactive proteins and the reciprocal concentration of each form in logarithmically growing cells suggest that the immunoreactive large species may be a precursor of the active enzyme.
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PMID:Identification of a high-molecular-weight nonfunctional protein in L1210 leukemia cells with common antigenic determinants to dihydrofolate reductase. 618 48

Dihydrofolate reductase has been purified from a trimethoprim-resistant strain of Neisseria gonorrhoeae. The enzyme showed a single component on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 18,000) and on isoelectric focusing in 5 M urea (pI = 6.8). Although gel electrophoresis under nondenaturing conditions resolved the preparation into two enzymatically active proteins (called form 1 and form 2), they were not genetically determined isozymes. Both had a similar dihydrofolate Km (2 microM), NADPH Km (10 microM), and trimethoprim Ki (20 nM), and form 2 (the slower migrating species) was shown to be generated from form 1 by the electrophoresis conditions. The complete covalent structure of the enzyme has also been determined. It is a single polypeptide composed of 162 residues and containing 4 cysteines. The gonococcal dihydrofolate reductase shares a 35% homology with the chicken liver enzyme and a 40% homology with the Escherichia coli enzyme. Most of these identities are residues that have been implicated in the binding of NADPH and methotrexate to the E. coli and Lactobacillus casei reductases.
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PMID:Characterization and amino acid sequence of Neisseria gonorrhoeae dihydrofolate reductase. 643 41

Dihydrofolate reductase isozyme 2 of Streptococcus faecium has been labeled with 13C in the C gamma position of tryptophan residues by growing the organism on a defined medium containing L-[gamma-13C]tryptophan (90% 13C). The 13C nuclear magnetic resonance (NMR) spectrum of the enzyme shows four well-resolved resonances which have nuclear Overhauser enhancements of 1.1-1.3. Values of T1 (spin-lattice relaxation time) and T2 (spin-spin relaxation time) are significantly less than predicted for an isotropically rotating, rigid sphere with no intermolecular dipole-dipole interactions. Three of the resonances have chemical shifts downfield from the 13C resonance of urea-denatured enzyme by amounts up to 1.43 ppm. The chemical shift of resonance 4 in the spectrum is 4.0 ppm upfield from Trp C gamma of urea-denatured enzyme. This large upfield shift is attributed to electric field effects generated by polar side chains. The two more upfield peaks both provide evidence that the corresponding tryptophan residues, WC and WD, each undergo chemical exchange between alternative microenvironments. In the case of WC, which gives a resonance with two components, exchange is slow (ve, exchange rate much less than 55 s-1), and the relative populations of the two stable states are in the ratio 2:3. WD is apparently in intermediate to fast exchange on the NMR time scale. With a two-state model, ve increases from approximately 90 to 150 s-1 as the temperature is increased from 5 to 25 degrees C. This increases in temperature is also accompanied by an increase in the fractional population of the minor downfield state(s), from about 0.062 at 5 degrees C to 0.24 at 25 degrees C. However, the data may also be interpreted as a temperature-dependent equilibrium between a continuum of many states. WD is tentatively identified with Trp-22 since comparison of the sequences of Lactobacillus casei dihydrofolate reductase and S. faecium dihydrofolate reductase and inspection of the crystal structure of the L. casei enzyme indicate that Trp-6, Trp-115, and Trp-160 are probably all involved in regions of beta sheet whereas Trp-22 is in a loop joining beta A to alpha B. Earlier crystallographic evidence for the Escherichia coli reductase suggests that in the methotrexate complex with this enzyme the corresponding loop has a good deal of flexibility. It is probable that in the uncomplexed S. faecium reductase the motion of this loop is the major mechanism for the exchange process involving Trp-22. The upfield chemical shift of resonance 4 is attributed to electric field effects on C gamma of Trp-22 produced by the carboxylate groups of Asp-27 and Asp-9. On the basis of the small difference between the chemical shift of resonance 3 and that of tryptophan C gamma in urea-denatured reductase, it is suggested that WC may be identified with Trp-6.
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PMID:Nuclear magnetic resonance study of dihydrofolate reductase labeled with [gamma-13C]tryptophan. 679 11


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