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)

The unambiguous synthesis of two folate analogues, in which the 10-amino group of folic acid was replaced with oxygen, is described. The synthetic sequence employed commercially available methyl p-hydroxybenzoate and n-(2,3-epoxypropyl)phthalimide as starting materials. The use of cesium bicarbonate as a coreactant in the nucleophilic displacement reaction between bromo ketone 3 and the nucleophile 4 was found to be unique in character. The aminoacetonyl oxime 7 obtained by the hydrazinolysis of 6 was used as a common intermediate for the synthesis of both compounds. The generality of the use of the TFA-HCL mixture to deprotect the carbonyl group of both 10 and 12 reductions involving sodium hydrosulfite in aqueous dmf were further substantiated by conversions of 11 and 13 to 14 and 15 quickly and efficiently without employing catalytic hydrogenations. Subsequent cyclizations, oxidations, and hydrolysis of these reduction products to the pteroate analogues 17 and 19 were carried out efficiently as described for the synthesis of the sulfur analogues. Activation of the carboxyl group of 19 by way of the mixed anhydride 22 and subsequent coupling to glutamic acid was carried out using the solid-phase coupling procedure. However, compound 17 required trifluoroacetylation to 20 prior to the coupling reaction due to solubility problems. Both 10-oxafolic acid (1) and 10-oxaaminopterin (2) showed potent antifolate activity when tested against two folate-requiring organisms. Compound 2 was a very powerful inhibitor of DCM-resistant lactobacillus casei dihydrofolate reductase. The activity was comparable to that of methotrexate while the 4-hydroxy analogue did not show inhibition. 7,8-Dihydro-10-oxafolic acid failed to show any substrate activity to this enzyme and did not inhibit the enzymatic reaction when used with an equimolar concentration of the natural substrate.
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PMID:Folate analogues altered in the C9-N10 bridge region. 10-Oxafolic acid and 10-oxaaminopterin. 82 Aug 58

The transit peptide of the lumenal 33-kDa oxygen-evolving polypeptide (OEE1) is capable of directing the import and targeting of the foreign protein dihydrofolate reductase (DHFR) to the thylakoid lumen. The import results from the first part of this study indicate that methotrexate cannot block the import or intraorganellar targeting of OEE1-DHFR in chloroplasts in contrast to that reported for the import of cytochrome oxidase subunit IV (COXIV)-DHFR in mitochondria. These results suggest that the fusion of the OEE1 transit sequence to DHFR affected the protein's methotrexate binding properties. We further examined and compared the transport characteristics of a number of carboxyl-terminal truncated native chloroplast precursors to determine whether carboxyl domains contribute to the import and intraorganellar targeting mechanism of these proteins. The plastid precursors chosen for this study are targeted to one of the following chloroplast compartments: the stroma, the thylakoid membrane, and the lumen. In most cases, removal of carboxyl domains had a dramatic effect on one or more stages of the translocation pathway, such as import, processing, and intraorganellar targeting. The effects of carboxyl deletions varied from precursor to precursor and were dependent on the extent of the deletion. These combined results suggest that carboxyl domains in the mature part of the proteins can influence the function of the transit peptide, and as a result play an important role in determining the import and targeting competence of chloroplast precursors.
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PMID:Carboxyl-terminal sequences can influence the in vitro import and intraorganellar targeting of chloroplast protein precursors. 132 Nov 29

Using site-specific mutagenesis, we have constructed two mutants of Escherichia coli dihydrofolate reductase (ecDHFR) to investigate further the function of a weakly acidic side chain at position 27 in substrate protonation: Asp27-->Glu (D27E) and Asp27-->Cys (D27C). The crystal structure of D27E ecDHFR in a binary complex with methotrexate shows that the side-chain oxygen atoms of Glu27 are in almost precisely the same location as those of Asp27 in the wild-type enzyme. Kinetic evidence indicates that Glu27 can indeed function efficiently in the proton relay to dihydrofolate. Even though vertebrate DHFRs all have a glutamic acid at the structurally equivalent position, the kinetic properties of Glu27 ecDHFR more closely resemble those of wild-type bacterial DHFRs than of vertebrate DHFRs. The D27C mutation produced an enzyme still capable of relaying a proton to dihydrofolate, but with the intrinsic pKa in its pH-activity profiles shifted upward to values characteristic of the more basic thiolate group. The crystal structure of the binary complex with methotrexate reveals two unexpected features: (1) the Cys27 sulfhydryl group does not point toward the pteridine-binding site, but the side chain of this residue is instead rotated 120 degrees to interact with a tyrosine side chain projecting from a neighboring beta-strand; (2) a bound ethanol molecule occupies a cavity adjacent to methotrexate. Ethanol is a component of the crystallization medium.
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PMID:Structure and function of alternative proton-relay mutants of dihydrofolate reductase. 135 37

A technique is described for the isolation and purification of intact, respiratory-competent mitochondria from Schizosaccharomyces pombe. The purified mitochondria are capable of oxidizing NADH and succinate as respiratory substrates, indicating the presence of succinate dehydrogenase and an NADH dehydrogenase located on the outer surface of the inner membrane. Mitochondria display good respiratory control with an ADP/O ratio of < 2. Respiratory activity is linearly dependent upon the redox poise of the quinone pool, suggesting the presence of an unbranched respiratory pathway to molecular oxygen. Immunogold labelling using antisera raised against mitochondrial HSP70 proteins (SSP1, SSC1 and PHSP1) from three different species, namely S. pombe, Saccharomyces cerevisiae and the plant Pisum sativum respectively, has been used to investigate the presence and ultrastructure of the mitochondria isolated by this procedure. The immunocytochemistry was carried out using cells containing wild-type levels of SSP1 protein and cells over-expressing the protein. These results also demonstrate the capacity of mitochondria to import increased levels of protein in vivo. In vitro import experiments using COXIV-DHFR indicate that purified S. pombe mitochondria can efficiently import this precursor, and that protein translocation is dependent upon an oxidizable substrate and a membrane potential.
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PMID:Schizosaccharomyces pombe mitochondria: morphological, respiratory and protein import characteristics. 148 70

The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.
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PMID:Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase. 161 Aug 17

The active sites of all bacterial and vertebrate dihydrofolate reductases that have been examined have a tryptophan residue near the binding sites for NADPH and dihydrofolate. In cases where the three-dimensional structure has been determined by X-ray crystallography, this conserved tryptophan residue makes hydrophobic and van der Waals interactions with the nicotinamide moiety of bound NADPH, and its indole nitrogen interacts with the C4 oxygen of bound folate through a bridge provided by a bound water molecule. We have addressed the question of why even the very conservative replacement of this tryptophan by phenylalanine does not seem to occur naturally. Human dihydrofolate reductase with this replacement of tryptophan by phenylalanine has increased rate constants for dissociation of substrates and products and a considerably decreased rate of hydride transfer. These cause some changes in steady-state kinetic behavior, including substantial increases in Michaelis constants for NADPH and dihydrofolate, but there is also a 3-fold increase in kcat. The branched mechanistic pathway for this enzyme has been completely defined and is sufficiently different from that of wild-type enzyme to cause changes in some transient-state kinetics. The most critical changes resulting from the amino acid substitution appear to be a 50% decrease in stability and a decrease in efficiency from 69% to 21% under intracellular conditions.
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PMID:Role of the conserved active site residue tryptophan-24 of human dihydrofolate reductase as revealed by mutagenesis. 199 Nov 24

The dissociation constants (pKa) for the pteridine ring system of dihydrofolate (H2folate) have been redetermined, and those for dihydrobiopterin (H2biopterin) have been determined. Determination of the pKa for N5 of H2folate is complicated by the low solubility and instability of H2folate at pH 2-4, and other complicating factors. The initial rate of absorbance change due to degradation is a maximum at pH 2.5, and the products depend on the oxygen concentration: under aerobic conditions, (p-aminobenzoyl)glutamic acid and 7,8-dihydropterin-6-carboxaldehyde are major products. H2Biopterin is much more soluble and more stable at low pH. For protonation of N5, the pKa is 2.56 +/- 0.01 for H2biopterin and 2.59 +/- 0.03 for H2folic acid. Spectrophotometric determination of the pKa for the N3-O4 amide group of H2folate is subject to serious errors when a wavelength between 220 and 235 nm is used. These errors arise from the pH-dependent absorbance of mercaptoethanol often present in the preparation. The amide group has a pKa of 10.41 +/- 0.04 in H2biopterin and 10.85 +/- 0.04 in H2folate. The redetermined value for the pKa of N5 of H2folate has implications for mechanistic models for dihydrofolate reductase, and revised kinetic constants have been calculated for one model.
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PMID:Dissociation constants for dihydrofolic acid and dihydrobiopterin and implications for mechanistic models for dihydrofolate reductase. 237 39

The HSITE program proposed in the previous paper was written to define putative ligand-point regions that could be found at protein surfaces. These regions would represent positions for hydrogen-bonding acceptor and donor atoms. In this paper the prediction of the location of these regions is compared with: (1) the position of the oxygen atoms of water molecules on the hydrated proteins myoglobin and plastocyanin; and (2) the position of hydrogen-bonded atoms in methotrexate and NADPH co-crystallized with dihydrofolate reductase, and in amidinophenyl-pyruvate co-crystallized with trypsin. The prediction of ligand-point regions is in agreement with the surveys of experimental data for water-molecule positions in protein crystals and with the positions of hydrogen-bonding atoms found in co-crystallized ligands.
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PMID:Automated site-directed drug design: the prediction and observation of ligand point positions at hydrogen-bonding regions on protein surfaces. 256 76

Giardia lamblia, an aerotolerant anaerobe, respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system. Glucose appears to be the only sugar catabolized by the Embden-Meyerhof-Parnas and hexose monophosphate pathways, and energy is produced by substrate level phosphorylation. Substrates are incompletely oxidized to CO2, ethanol and acetate by nonsedimentable enzymes. The lack of incorporation of inosine, hypoxanthine, xanthine, formate or glycine into nucleotides indicates an absence of de novo purine synthesis. Only adenine, adenosine, guanine and guanosine are salvaged, and no interconversion of these purines was detected. Salvage of these purines and their nucleosides is accomplished by adenine phosphoribosyltransferase, adenosine hydrolase, guanosine phosphoribosyltransferase and guanine hydrolase. The absence of de novo pyrimidine synthesis was confirmed by the lack of incorporation of bicarbonate, orotate and aspartate into nucleotides, and by the lack of detectable levels of the enzymes of de novo pyrimidine synthesis. Salvage appears to be accomplished by the action of uracil phosphoribosyltransferase, uridine hydrolase, uridine phosphotransferase, cytidine deaminase, cytidine hydrolase, cytosine phosphoribosyltransferase and thymidine phosphotransferase. Nucleotides of uracil may be converted to nucleotides of cytosine by cytidine triphosphate synthetase, but thymidylate synthetase and dihydrofolate reductase activities were not detected. Uptake of pyrmidine nucleosides, and perhaps pyrimidines, appears to be accomplished by carrier-mediated transport, and the common site for uptake of uridine and cytidine is distinct from the site for thymidine. Thymine does not appear to be incorporated into nucleotide pools. Giardia trophozoites appear to rely on preformed lipids rather than synthesizing them de novo.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemistry and metabolism of Giardia. 265 35

In a semi-defined minimal medium for cultivation of Plasmodium falciparum, ribose, mannose, fructose, galactose, and maltose could not replace glucose. Hypoxanthine was the preferred purine source for the parasite over adenine, guanine, inosine, adenosine and guanosine although all supported growth equally. Inhibitors of nucleoside uptake had low potency in killing the parasites but depressed incorporation of [3H]adenosine more than [3H]hypoxanthine. Glutamate could not be replaced by 5-oxoproline, indicating that the gamma-glutamyl transferase pathway for amino acid uptake is probably not found in this organism. Adenine, nicotinamide, and orotic acid could not supplement glutamine-deficient medium. The pyridoxine antagonists isoniazid and 4-deoxypyridoxine were reversed by amino acid supplementation, suggesting that transaminases may be targets of these drugs. Orotic acid, but not glutathione or its amino acid components, partially reversed the effects of 8-methylamino-8-desmethyl riboflavin. Thus, the flavin enzyme, dihydroorotic acid dehydrogenase, but not glutathione reductase, appears to be a target of this riboflavin antagonist. Five biotin antagonists had no significant activity. The choline antagonist 2-(tert-butylamino)ethanol and thiamin uptake inhibitors had nonspecific inhibitory effects, which were not reversed by the respective target vitamin. Buthionine sulfoximine and methionine sulfoximine, inhibitors of glutathione synthesis, had significant oxygen-dependent toxicity. Six sulfonamides showed marked variation in potency and efficacy. Sulfathiazole and sulfadoxine were reversed differentially by p-aminobenzoic acid, folic acid, and folinic acid. Folinic acid was more effective than folic acid at reversing the toxicity of the dihydrofolate reductase inhibitors aminopterin and pyrimethamine; p-amino-benzoic acid had no effect.
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PMID:Nutritional requirements of Plasmodium falciparum in culture. III. Further observations on essential nutrients and antimetabolites. 286 44


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