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 role of eukaryotic initiation factor 2 (eIF-2) phosphorylation in translational control has been demonstrated in vivo by overexpressing variant forms of eIF-2 alpha that are not phosphorylated. COS-1 cells transiently transfected with expression vectors for human eIF-2 alpha contain 10-20-fold more eIF-2 alpha subunit than the endogenous COS cell eIF-2 trimeric complex. Expression of the variant form of eIF-2 alpha, Ser51Asp, where Asp replaces Ser51, causes inhibition of protein synthesis, whereas the Ser48Asp variant does not. When either Ser48 or Ser51 is replaced by Ala, the variants stimulate dihydrofolate reductase synthesis when the eIF-2 alpha kinase, DAI, is activated. In order to elucidate these mechanisms, we have separated eIF-2 trimeric complexes from free overexpressed eIF-2 alpha subunits by fast protein liquid chromatography Superose chromatography. Pulse-labeled cells transfected with wild-type or variant DNAs produced eIF-2 preparations with greater than 10-fold higher specific radioactivity in the alpha-subunit compared to the gamma-subunit, thus demonstrating that the human eIF-2 alpha produced from the plasmids readily exchanges into COS cell eIF-2 complexes. Both wild-type and Ser48Ala variant forms of the free 2 alpha-subunit, further purified by MonoQ chromatography, are poor substrates for the heme-regulated eIF-2 alpha kinase, HRI, but are good substrates for double-stranded RNA-activated inhibitor in vitro; the Ser51Ala variant subunit is not phosphorylated by either kinase. None of the purified free eIF-2 alpha subunits inhibits phosphorylation of eIF-2 in vitro, even at up to 8-fold molar excess. Examination of the extent of eIF-2 alpha phosphorylation in the COS cell eIF-2 complexes by two-dimensional polyacrylamide gel electrophoresis shows that the stimulation of dihydrofolate reductase synthesis by the Ser51Ala variant is most readily explained by failure of eIF-2 to be phosphorylated. Stimulation by the Ser48Ala variant appears to occur by mitigation of the effect of phosphorylation at Ser51 since the double variant, Ser48Ala-Ser51Asp, inhibits protein synthesis less than the single variant Ser51Asp. The evidence argues strongly against there being a second site of phosphorylation involved in translational repression.
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PMID:Stimulation of protein synthesis in COS cells transfected with variants of the alpha-subunit of initiation factor eIF-2. 134 29

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.
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PMID:Cloning and expression of a human ATP-citrate lyase cDNA. 137 49

The middle base (U35) of the anticodon of tRNA(Gln) is a major element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). An opal suppressor of tRNA(Gln) (su+2UGA) containing C35 (anticodon UCA) was isolated by genetic selection and mutagenesis. Suppression of a UGA mutation in the E. coli fol gene followed by N-terminal sequence analysis of purified dihydrofolate reductase showed that this tRNA was an efficient suppressor that inserted predominantly tryptophan. Mutations of the 3-70 base pair (U70 and A3U70) were made. These mutants of su+2UGA are less efficient suppressors and inserted predominantly tryptophan in vivo; alanine insertion was not observed. Mutations of the discriminator nucleotide (A73, U73, C73) result in very weak opal suppressors. Aminoacylation in vitro by E. coli TrpRS of tRNA(Gln) transcripts mutated in the anticodon demonstrate that TrpRS recognizes all three nucleotides of the anticodon. The results show the interchangeability of the glutamine and tryptophan identities by base substitutions in their respective tRNAs. The amber suppressor (anticodon CUA) tRNA(Trp) was known previously to insert predominantly glutamine. We show that the opal suppressor (anticodon UCA) tRNA(Gln) inserts mainly tryptophan. Discrimination by these synthetases for tRNA includes position 35, with recognition of C35 by TrpRS and U35 by GlnRS. As the use of the UGA codon as tryptophan in mycoplasma and in yeast mitochondria is conserved, recognition of the UCA anticodon by TrpRS may also be maintained in evolution.
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PMID:Switching tRNA(Gln) identity from glutamine to tryptophan. 156 39

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

Most antibodies directed against the Plasmodium falciparum circumsporozoite (CS) protein react with its central domain, which contains about 40 repeats of the tetrapeptide Asn-Ala-Asn-Pro (NANP). To search for new epitopes in the non-repetitive part of the CS protein, we expressed the non-repetitive regions of the protein in E. coli as fusion proteins with mouse dihydrofolate reductase linked to six adjacent histidine residues. These fusion proteins were obtained at greater than 70% purity by a single Ni-chelate affinity chromatography step. Of the new epitopes defined in the C-terminal portion of the CS protein, three are located in a stretch of 65 amino acids immediately C-terminal of the protein's central repetitive domain. Pooled sera from inhabitants of a malaria-endemic area reacted with epitopes in this region of the molecule, and four mouse monoclonal antibodies to this region also reacted with the native CS protein on sporozoites. Two of the monoclonal antibodies reacted with a peptide PNDPNRNVD derived from a conserved region of the CS protein. The other two antibodies showed different reactivities to sporozoites of the NF54 and Ro59 parasite isolates. One, which reacted with a peptide ENANANNAV, recognized Ro59 but not NF54 sporozoites, while the other reacted with a small percentage of NF54 but not Ro59 sporozoites. Antibodies which react with non-repetitive regions of the CS protein could contribute to maintaining its genetic variability.
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PMID:New B cell epitopes in the Plasmodium falciparum malaria circumsporozoite protein. 169 36

The importance of three amino acid residues contacting the nicotinamide ring of NADPH in Escherichia coli dihydrofolate reductase has been defined using site-directed mutagenesis and detailed steady-state and pre-steady-state kinetic experiments. Replacement of Tyr-100 with either glycine or isoleucine (Y100G or Y100I) disrupts an aromatic-aromatic interaction between the phenolic side chain and the nicotinamide ring. Both mutations remove the differential binding of the oxidized and reduced coenzymes implicating Tyr-100 as a major determinant for coenzyme specificity. Replacement of Ser-49 for alanine (S49A), designed to either displace or reduce the polarizability of a bound water molecule contacting the N1 of the nicotinamide ring, affects only the rate of release of NADP+. Replacement of Ile-14 with alanine (I14A), designed to alter both a weakly polar and a hydrogen bonding interaction with the periphery of the nicotinamide ring, affects only the binding of NADPH. Y100I, Y100G, and I14A all increase the activation barrier for the chemical step by approximately 2 kcal/mol. The lack of an effect for S49A suggests that water structure is not important for stabilizing the hydride transfer transition state. In addition, the nominal effects observed for these mutations disfavor the hypothesis that neighboring amino acid residues participate in the stabilization of the reaction transition state through polar or weakly polar contacts.
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PMID:The function of amino acid residues contacting the nicotinamide ring of NADPH in dihydrofolate reductase from Escherichia coli. 183 73

The role of a hinge region in the folding, stability, and activity of Escherichia coli dihydrofolate reductase was investigated with three site-directed mutants at valine-88, the central residue of the hinge. The three mutants, V88A and V88I and a valine-88 deletion, were created to perturb the packing of hydrophobic residues in the interior of a loose turn formed by residues 85-91. Deleting the valine-88 residue destabilized the protein by 2.93 +/- 0.6 kcal/mol as determined by equilibrium unfolding transitions in urea monitored by circular dichroism at 20 degrees C. Substitution of alanine for valine-88 stabilized the protein by -0.20 +/- 0.02 kcal/mol, and the isoleucine substitution was mildly destabilizing by 1.73 +/- 0.2 kcal/mol. Although there was no clear correlation between side-chain volume and stability, these results suggest that side-chain interactions in the interior of the turn influence the folding and stability of dihydrofolate reductase. The specific activity of the valine deletion mutant was approximately twice that of the wild-type protein while the specific activities of the V88A and V88I proteins were only slightly greater than the wild type. The full time courses of the reactions catalyzed by the mutants were almost identical with that for the wild type, indicating no major changes in the kinetic mechanism. Additionally, the rate constants associated with interconversion between various forms of the apoenzyme were identical for the mutant and wild-type enzymes. The rate constants for refolding transitions were examined by dilution of urea-inactivated protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of point mutations in a hinge region on the stability, folding, and enzymatic activity of Escherichia coli dihydrofolate reductase. 186 58

Cycloguanil, the active metabolite of the antimalarial drug proguanil, is an inhibitor of dihydrofolate reductase as is another antimalarial, pyrimethamine. Its use has been limited by the rapid development of resistance by parasites around the world. We have determined the cycloguanil- and pyrimethamine-sensitivity status of 10 isolates of Plasmodium falciparum and have sequenced in all these isolates the dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) portion of the DHFR-thymidylate synthase (TS; 5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) gene. Instead of the known serine-to-asparagine change at position 108 that is important in pyrimethamine resistance, a serine-to-threonine change at the same position is found in cycloguanil-resistant isolates along with an alanine-to-valine change at position 16. We conclude that pyrimethamine and cycloguanil resistance most commonly involve alternative mutations at the same site. However, we also have identified a parasite with a unique set of changes that results in resistance to both drugs.
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PMID:Amino acids in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum involved in cycloguanil resistance differ from those involved in pyrimethamine resistance. 218 21

Proguanil and pyrimethamine are antifolate drugs with distinct chemical structures that are used commonly in the prophylaxis and treatment of Plasmodium falciparum malaria. Clinical reports and field studies have suggested that some parasites refractory to proguanil can be treated with pyrimethamine, and vice versa. Analysis of the P. falciparum dihydrofolate reductase (DHFR) from different parasites reveals the structural basis for differential susceptibility to these antifolate drugs. Parasites harboring a pair of point mutations from Ala-16 to Val-16 and from Ser-108 to Thr-108 are resistant to cycloguanil (the active metabolite of proguanil) but not to pyrimethamine. A single Asn-108 mutation, on the other hand, confers resistance to pyrimethamine with only a moderate decrease in susceptibility to cycloguanil. Significant cross-resistance to both drugs occurs in parasites having mutations that include Ser-108----Asn-108 and Ile-164----Leu-164. These results reflect the distinct structures of pyrimethamine and cycloguanil and suggest fine differences in binding within the active site cavity of DHFR. Alternative inhibitors, used alone or in combination, may be effective against some strains of cycloguanil- or pyrimethamine-resistant malaria.
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PMID:Molecular basis of differential resistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria. 218 22

We have studied the specificity requirements for processing of the human insulin proreceptor by successively replacing each basic amino acid in the tetrabasic cleavage site with alanine. These mutated receptor cDNAs have then been overexpressed in Chinese hamster ovary cells, using vectors containing the mouse dihydrofolate reductase gene to amplify the transfected cDNAs in the presence of increasing concentrations of methotrexate. High levels of expression, ranging up to 6 x 10(7) receptors/cell were achieved in these experiments. Replacement of the P1 arginine with alanine led to the complete suppression of processing, as occurs also in a naturally occurring serine mutation at this site (Yoshimasa, Y., Seino, S., Whittaker, J., Kakehi, T., Kosaki, A., Kuzuya, H., Imura, H., Bell, G. I., and Steiner, D. F. (1988) Science 240, 783-787). A small amount of cleavage at alternative sites was detected. Replacement of the P2 arginine or P3 lysine with alanine did not in either case affect conversion to mature alpha and beta subunits, while replacement of the P4 arginine significantly inhibited processing. The binding isotherms for the processed versions of the receptor were comparable to previously published normal values. The unprocessed proreceptor bound insulin normally but was autophosphorylated less efficiently than processed versions of the receptor expressed in the same cells. These results suggest that a single processing protease with trypsin-like specificity may be involved in processing both insulin and insulin-like growth factor-I receptor precursors as well as a variety of viral envelope glycoprotein precursors.
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PMID:Effects of amino acid replacements within the tetrabasic cleavage site on the processing of the human insulin receptor precursor expressed in Chinese hamster ovary cells. 221 23


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