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)

N alpha-(4-Amino-4-deoxy-N10-methylpteroyl)-N epsilon-(iodoacetyl)-L-lysine (1) was synthesized as a potential active-site-directed irreversible inhibitor of dihydrofolate reductase (DHFR). In an ultraviolet spectrophotometric assay of dihydrofolate reduction of Lactobacillus casei DHFR, 1 and methotrexate (MTX, 4-amino-4-deoxy-N10-methylpteroyl-L-glutamic acid) had ID50 values of 4.5 and 6.2 nM. The corresponding ID50 values in a competitive radioligand binding assay against [3H]MTX were 31 and 16 nM. Thus, as reversible inhibitors of this enzyme over a short exposure time, 1 and MTX had comparable activity. On the other hand, when L. casei DHFR was incubated for up to 6 h with 0.1 or 1.0 microM 1, a progressive decrease in the ability of [3H]MTX to subsequently displace the drug was observed. When MTX itself was used at the same concentrations, the extent of displacement of [3H]MTX did not decrease with time. These results were consistent with rapid reversible binding of 1 to the enzyme, followed more slowly by covalent bond formation near the active site. The pH profile for this effect followed a curve with a sigmoidal shape. The apparent inflection point near pH 7.2 was consistent with alkylation of a histidine residue.
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PMID:Methotrexate analogues. 15. A methotrexate analogue designed for active-site-directed irreversible inactivation of dihydrofolate reductase. 681 44

Methotrexate (MTX) conjugated to a Mr 3000 poly(L-Lys) markedly inhibits the growth of Pro-3 MtxRII5-3 Chinese hamster ovarian cells, a mutant line known to be drug resistant because of defective MTX transport. In these cells, membrane transport of [3H]MTX-poly(Lys) is sharply decreased by addition of 0.5- to 2.5-fold heparin but remains at 15-20% of control in 2.5- to 50-fold heparin excess. Heparin addition at first markedly inhibits but, at high concentration, restores the growth inhibitory effect of MTX-poly(Lys). In excess heparin, MTX-poly(Lys) is transported as a heparin complex. Because reduced transport (15-20%) is sufficient to cause a 90% inhibition of cell growth, MTX-poly(Lys) apparently gains pharmacologic potency when compared to heparin. This gain can be related to a greater inhibitory effect on dihydrofolate reductase and to a different mode of transport. The inhibitory effect of MTX-poly(Lys) on dihydrofolate reductase in vitro is increased nearly 100-fold in the presence of excess heparin but remains less than that of free MTX. Unlike that of MTX-poly(Lys), the transport of MTX-poly(Lys)-heparin has the characteristics and efficiency of a receptor-mediated process. It proceeds by endocytosis but is not, as in the case of uncomplexed conjugate, followed by the intracellular generation of pharmacologically active breakdown products that would account for cytotoxicity. These observations raise the possibility that at least part of the MTX-poly(Lys)-heparin reaches cellular dihydrofolate reductase in the form of macromolecular complexes that escape from entrapment in endocytotic structures. Our data illustrate a way to overcome drug resistance by taking advantage of the specific uptake of a macromolecular drug carrier. They offer a method of drug delivery in which heparin improves selectivity and decreases the unwanted toxicity inherent to polycationic carriers.
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PMID:Poly(L-lysine) has different membrane transport and drug-carrier properties when complexed with heparin. 695 Apr

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

The ornithine (6a) and lysine (6b) analogues of methotrexate (1) have been synthesized via condensation of 4-amino-4-deoxy-N10-methylpteroic acid (2) with N gamma-carbobenzoxy-L-ornithine tert-butyl ester (3a) and N epsilon-carbobenzoxy-L-lysine tert-butyl ester (3b), respectively. Removal of the protecting groups gave 5a and 6b. Compounds 6a and 6b and their precursor Cbz acids (5a and 5b) show significant inhibition of dihydrofolate reductase.
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PMID:Lysine and ornithine analogues of methotrexate as inhibitors of dihydrofolate reductase. 706 26

A new fluorescent methotrexate analogue (PT430) was synthesized as a reported ligand for dihydrofolate reductase. The analogue was prepared by attachment of lysine in place of the glutamate side chain of methotrexate and conjugation to fluorescein isothiocyanate via the epsilon-amino group of lysine. Spectrophotometric enzyme inhibition assays showed PT430 to be about one-tenth as potent as methotrexate against either Lactobacillus casei or L1210 mouse leukemia enzyme; competitive radioligand binding assays using tritiated methotrexate gave similar results. In assays of L1210 cell proliferation in culture, on the other hand, PT430 was 100-fold less toxic than methotrexate. In dilute solution, the fluorescence intensity of PT430 was 5-fold lower than that of equimolar fluorescein and diminished with decreasing pH. On complexation with dihydrofolate reductase, however, fluorescence intensity was enhanced 3- to 5-fold depending on the pH. Measurement of fluorescence increase with added ligand provided data for the determination of the stoichiometric ratio, dissociation constant, and extent of fluorescence enhancement. Specificity of PT430 for methotrexate binding sites was indicated by the observation of decreased fluorescence uptake in PT430-treated L1210 cells in the presence of methotrexate. Fluorescence uptake occurred faster, and to a greater extent, in methotrexate-resistant dihydrofolate reductase overproducing L1210/R6 cells than in the methotrexate-sensitive parent line. Therefore, PT430 may be used as a flow cytometry probe to detect methotrexate resistance based on dihydrofolate reductase overproduction.
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PMID:A new fluorescent dihydrofolate reductase probe for studies of methotrexate resistance. 714

The variation with pH of the kinetic parameters associated with dihydrofolate reductase from Streptococcus faecium has been used to gain information about the chemical mechanism of the reaction catalyzed by the enzyme. The pH dependence of log V/K for dihydrofolate showed that a group with a pK value of 4.7 must be ionized and that a group with a pK value of 6.6 must be protonated for activity. Temperature and solvent perturbation studies indicate that these groups are probably the carboxyls of the glutamate moiety of dihydrofolate and of an aspartate residue on the enzyme, respectively. The similarity of the pH profile and the magnitude of the pK value for the linear competitive inhibitor 2,4-diaminopteridine suggest that the carboxyl group is concerned with the binding of dihydrofolate and its analogues to the enzyme. This conclusion is confirmed by the result that a group with a pK value of 6.7 must be protonated for the binding of methotrexate. It is proposed that the binding involves the formation with N-5 of dihydrofolate or N-1 of methotrexate of a hydrogen bond which has considerable ionic character and which lies within a hydrophobic environment. Further, it is suggested that the same hydrogen acts as an auxiliary catalyst which facilitates hydride transfer from NADPH to dihydrofolate for its conversion to tetrahydrofolate. Evidence to support this suggestion comes from the finding that the V profile is similar to the V/K profile except that the pK of the group which must be protonated for maximum enzyme activity is shifted upward by about 2 pH units. Such an increase in a pK value is consistent with the formation of a hydrogen ionic bond in the ternary enzyme-NADPH-dihydrofolate complex. The results of inactivation experiments with trinitrobenzenesulfonate appear to indicate that a lysine residue is necessary to maintain the enzyme in its active conformation.
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PMID:Chemical mechanism of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium: pH studies and chemical modification. 730 91

Methotrexate (MTX) was conjugated through a carbodiimide-catalyzed reaction to poly (L-lysines) of various molecular sizes at a ratio of approximately one molecule per 27 lysyl residues. These conjugates were tested on cultured, Chinese hamster, ovary cells known to be drug resistant because of a deficient methotrexate transport. The cellular uptake of conjugated drug far exceeded the uptake of free drug in both drug sensitive and resistant lines. The conjugated drug inhibited the growth of the transport-deficient cells at concentrations at which free drug had no effect. The conjugate failed to inhibit dihydrofolate reductase in vitro. This and other evidence indicate that the strong pharmacologic effect of the MTX-poly(Lys) conjugate is due to the intracellular--presumably intralysosomal--hydrolysis of its polymeric backbone followed by the release inside the cell of an active form of MTX. This conclusion is supported by data obtained with conjugates using poly(D-lysine) as a carrier. This optical isomer is not susceptible to common proteolytic enzymes and MTX-poly(D-Lys) has not growth inhibitory effect whatever on either transport proficient or deficient CHO-cells. MTX-poly(L-lys) can thus be seen as a lysosome-activated drug.
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PMID:Conjugation of methotrexate to poly (L-lysine) as a potential way to overcome drug resistance. 735 12

Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.
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PMID:A proteolytic pathway that recognizes ubiquitin as a degradation signal. 761 50

A new method for assaying ubiquitin C-terminal hydrolases was developed using a 125I-labeled ubiquitin-alpha NH-MHISPPEPESEEEEEHYC was substrate. Since the peptide portion was almost exclusively radiolabeled, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid-soluble products. Using this assay protocol, we identified at least 10 ubiquitin C-terminal hydrolase activities from the extract of chick skeletal muscle, which were tentatively named UCHs 1 through 10. Of these, UCH-6 was purified to apparent homogeneity. Purified UCH-6 behaved as a dimer of 27-kDa subunits. The apparent molecular masses of the other partially purified UCHs ranged from 35 to 810 kDa as determined under a non-denaturing condition. Muscle UCHs, except UCH-1, were activated dramatically by poly-L-Lys but with an unknown mechanism. All of the UCHs were sensitive to inhibition by sulfhydryl-blocking agents such as iodoacetamide. In addition, all of the UCHs were capable of releasing free ubiquitin from a ubiquitin-alpha NH-carboxyl extension protein of 80 amino acids and from ubiquitin-alpha NH-dihydrofolate reductase. Five of the enzymes, UCHs 1 through 5, were also capable of generating free ubiquitin from poly-His-tagged diubiquitin. In addition, UCH-1 and UCH-7 could remove ubiquitin that had been ligated covalently by an isopeptide linkage to a ubiquitin (RGA)-alpha NH-peptide, the peptide portion of which consists of the 20 amino acids of the calmodulin binding domain of myosin light chain kinase. These results suggest that the 10 UCH activities isolated from chick skeletal muscle appear to be distinct from each other at least in their chromatographic behavior, size, and substrate specificity.
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PMID:Multiple ubiquitin C-terminal hydrolases from chick skeletal muscle. 764 26

The amino acid composition of proteins from mesophilic and extremophilic organisms is commonly assumed to reflect the mechanisms of molecular adaptation to extremes of physical conditions. In this context, halophilic behaviour has been attributed to significantly increased numbers of aspartic and glutamic acid residues. However, extending the analysis to a statistically relevant set of related proteins, dihydrofolate reductase from Halobacterium volcanii, as an example, shows that the increase in negative charge is found to be less significant than other exchanges of amino acids (e.g., Ala, Asn, Arg, Lys, Phe, Ser). Thus, the high water binding capacity of negatively charged residues cannot be unambiguously correlated with the anomalous stability of halophilic proteins. A similar caveat holds for generalizations regarding the thermal stability of proteins. In this case, D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was compared with a number of mesophilic and moderately thermophilic homologs. Again, 'traffic rules of stabilization', in terms of amino acid changes in going from mesophilic to thermophilic proteins, cannot be given.
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PMID:Relevance of sequence statistics for the properties of extremophilic proteins. 790 11


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