Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structure of the Escherichia coli thymidylate synthase (TS) covalent inhibitory ternary complex consisting of enzyme, 5-fluoro-2'-deoxyuridylate (FdUMP) and 5,10-methylene tetrahydrofolate (CH2-H4PteGlu) has been determined at 2.5 A resolution using difference Fourier methods. This complex is believed to be a stable structural analog of a true catalytic intermediate. Knowledge of its three-dimensional structure and that for the apo enzyme, also reported here, suggests for the first time how TS may activate dUMP and CH2-H4PteGlu leading to formation of the intermediate and offers additional support for the hypothesis that the substrate and cofactor are linked by a methylene bridge between C-5 of the substrate nucleotide and N-5 of the cofactor. By correlating these structural results with the known stereospecificity of the TS-catalyzed reaction it can be inferred that the catalytic intermediate, once formed, must undergo a conformational isomerization before eliminating across the bond linking C-5 of dUMP to C-11 of the cofactor. The elimination itself may be catalyzed by proton transfer to the cofactor's 5 nitrogen from invariant Asp169 buried deep in the TS active site. The juxtaposition of Asp169 and bound tetrahydrofolate in TS is remarkably reminiscent of binding geometry found in dihydrofolate reductase where a similarly conserved carboxyl group serves as a general acid for protonating the corresponding pyrazine ring nitrogen of dihydrofolate.
J Mol Biol 1990 Aug 20
PMID:Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate. 220 79

The action of 10-deazaaminopterin, its 10-alkyl derivatives, and their polyglutamates against thymidylate synthase (TMPS) from human acute myeloblastic leukemia was examined. Comparison of aminopterin with methotrexate showed that the methylation of the N10-position (methotrexate) increased the inhibitory effect of aminopterin on TMPS. In contrast, alkylation of the 10-position of 10-deazaaminopterin decreased inhibition of TMPS, and the 50% inhibitory concentration values were progressively higher, in the order 10,10-dimethyl-, 10-methyl-, and 10-ethyl-derivatives. The addition of gamma-glutamyl moieties to both 10-deazaaminopterin, and one of its alkylated analogs, 10-ethyl-10-deazaaminopterin, enhanced inhibition. The maximum inhibition was achieved with the addition of three glutamyl moieties to 10-deazaaminopterin and two glutamyl moieties to 10-ethyl-10-deazaaminopterin, respectively. Thus, 10-deazaaminopterin-tetraglutamate was 138-fold and 10-ethyl-10-deazaaminopterin-triglutamate was greater than 51-fold more active than their respective parental compound. The compounds 10-deazaaminopterin and its polyglutamates, 10-methyl- and 10,10-dimethyl-analogs, inhibited TMPS in a noncompetitive fashion with respect to 5,10-methylene-tetrahydropteroylglutamate. Ki values for the monoglutamates were 220 microM, 310 microM, and 225 microM, respectively. In contrast, 10-ethyl-10-deazaaminopterin and its polyglutamates inhibited TMPS in a competitive fashion with a Ki value of 410 microM for the monoglutamate. With 5,10-methylene-tetrahydropteroylpentaglutamate as a substrate, 10-deazaaminopterin and its polyglutamates behaved as mixed type inhibitors, and 10-ethyl-10-deazaaminopterin, monoglutamate and diglutamate, behaved as noncompetitive inhibitors, whereas its pentaglutamate behaved as a mixed-type inhibitor. These results suggest that the addition of gamma-glutamyl moieties to the substrate also caused the change in the mode of inhibitory action of these compounds. These findings also show that both replacement of the N10-position of the 4-aminopteroyl structure with a methylene group and its alkylation caused interesting and unexpected changes in the structure-activity relationships and the mode of action for these 4-aminopteroyl antifolates as inhibitors of TMPS, which may be therapeutically relevant.
Mol Pharmacol 1986 Aug
PMID:Inhibitory action of 10-deazaaminopterins and their polyglutamates on human thymidylate synthase. 242 68

Serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified from a pyrimethamine sensitive strain of Plasmodium chabaudi. Km values of 2.91 and 1.08 mM were determined for tetrahydrofolate and serine, respectively. The effects of pH, of temperature and of some potential inhibitors were determined. The enzyme was also partially purified from a pyrimethamine-resistant strain of P. chabaudi and subjected to the same regime. No differences between the enzymes from the two sources could be detected. It would appear that the changes in properties in the enzymes dihydrofolate reductase and thymidylate synthetase associated with the development of drug resistance in P. chabaudi were not reflected in any obvious alterations in serine hydroxymethyltransferase.
Mol Biochem Parasitol 1989 Mar 15
PMID:Serine hydroxymethyltransferase from pyrimethamine-sensitive and -resistant strains of Plasmodium chabaudi. 249 46

We have constructed a plasmid, pQS1, in which a mouse dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP:oxidoreductase; EC 1.5.1.3; DHFR) cDNA is inserted in the unique PstI site of a gram-positive/gram-negative shuttle vector derived from pBR322. The cDNA is expressed under the control of the bla promoter, which, like most gram-negative bacterial genes, is considered not to be expressed in Bacillus subtilis, and its coding sequence is translated from a polycistronic message. We have selected in vivo and studied, in Escherichia coli and B. subtilis, expression mutants with promoter and ribosome binding site sequence mutations. One promoter mutation changes the third nucleotide of the -35 region from a C to a G. As expected, this substitution results in increased transcriptional activity in E. coli. In B. subtilis, this mutation induces the accumulation not only of a low but significant amount of dhfr mRNA but also of DHFR, demonstrating that binding strengths with a free energy as low as -9.4 kcal/mol are sufficient to promote ribosome binding in B. subtilis. The association of the promoter mutation (C-G) with a mutation which creates a strong B. subtilis ribosome binding site (-21 kcal/mol) results in the accumulation of a large amount of dhfr mRNA. This demonstrates the importance of having an efficient ribosome binding site in the evaluation of promoter function: for example, with this strong ribosome binding site we can show that the wild-type bla promoter is recognized by the B. subtilis transcription machinery.
Mol Gen Genet 1989 Oct
PMID:In vivo selected promoter and ribosome binding site up-mutations: demonstration that the Escherichia coli bla promoter and a Shine-Dalgarno region with low complementarity to the 16 S ribosomal RNA function in Bacillus subtilis. 251 27

Methanol poisoning in primates and humans is due to formate accumulation as a result of low rates of formate oxidation. This toxicity is not seen in rats, where formate oxidation rates are high. Formate oxidation in vivo is dependent on hepatic tetrahydrofolate levels and on the activity of the enzyme 10-formyl-tetrahydrofolate (10-formyl-H4folate) dehydrogenase (EC 1.5.1.6). Because hepatic 10-formyl-H4folate dehydrogenase activity is lower in human liver than in rat liver, studies were performed investigating the properties of this enzyme in rat and human liver. 10-Formyl-H4folate dehydrogenase was purified to homogeneity from rat and human liver and was found to possess similar subunit molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (96,000). N-Terminal amino acid analysis of the pure proteins showed an identical sequence for the first 16 amino acids. Antibodies raised in rabbits against the rat liver enzyme were inhibitory toward the activity of both rat and human liver enzymes and appeared to recognize only the 10-formyl-H4folate dehydrogenase in cytosolic preparations of rat and human liver. Immunoblots of pure rat and human liver 10-formyl-H4folate dehydrogenase showed similar staining intensity. It is concluded that rat and human liver 10-formyl-H4folate dehydrogenase possess very similar properties and that the activity of the enzyme in human liver is lower than that of rat liver, due to a reduced amount of enzyme protein in human liver. This may be an important factor in regulating formate oxidation in humans and may explain, in part, the accumulation of formate and the mechanism of toxicity of methanol in humans.
Mol Pharmacol 1989 Jun
PMID:Studies on the mechanism of methanol poisoning: purification and comparison of rat and human liver 10-formyltetrahydrofolate dehydrogenase. 273 92

Studies have been performed in rats in order to test whether methionine reverses the inhibition of formate oxidation produced by nitrous oxide by virtue of the conversion of methionine to formate. At a dose of methionine (100 mg/kg, 671 mumol/kg) that completely reverses the nitrous oxide inhibition of formate oxidation no significant conversion of the methyl group, carboxyl, or backbone of methionine to formate was apparent. No increases in hepatic formate levels were seen after the administration of 671 mumol/kg methionine or ethionine, and formate treatment did not alter the rate of 14CO2 formed after methionine was administered labeled in the methyl, carboxyl, or backbone position. The reversal of nitrous oxide inhibition of formate oxidation was found to correlate temporally with either S-adenosylmethionine levels after methionine administration or S-adenosylethionine levels following ethionine treatment. After methionine or ethionine administration, elevated hepatic steady state levels of tetrahydrofolate were observed and were coincident with elevated S-adenosylmethionine or S-adenosylethionine. Since formate oxidation rates are dependent on the hepatic tetrahydrofolate level, the mechanism of methionine reversal of nitrous oxide inhibition appears to be related to effects of hepatic S-adenosylmethionine which are important in maintaining and regulating tetrahydrofolate, rather than formate generation from methionine.
Mol Pharmacol 1987 Aug
PMID:The role of formate and S-adenosylmethionine in the reversal of nitrous oxide inhibition of formate oxidation in the rat. 311 58

Dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADPH+-oxidoreductase; EC 1.5.1.3) was partially purified by affinity chromatography from three clones of the human malaria parasite Plasmodium falciparum. The three clones were representative of pyrimethamine-sensitive (clone 3D7) and pyrimethamine-resistant (clone HB3 and clone 7G8) parasites with ID50 values of 0.53 nM (3D7), 210 nM (HB3), and 540 nM (7G8), when tested in vitro against the drug. The specific activities of the partially purified DHFR differed by less than a factor of 2 between the sensitive clone 3D7 (442 +/- 39 nmol min-1 mg-1 protein) and the resistant clones HB3 (634 +/- 25 nmol min-1 mg-1 protein) and 7G8 (565 +/- 85 nmol min-1 mg-1 protein). The number of catalytic sites in partially purified DHFR from the three clones was similar and ranged from 151 to 194 pmol mg-1 protein. The Km value for NADPH was similar in all three clones (4.5-11.6 microM). The Km value for dihydrofolate was altered 13-fold comparing the sensitive clone 3D7 (3.2 +/- 0.6 microM) with the resistant clone HB3 (42.6 +/- 1.6 microM), with the Km for the resistant clone 7G8 falling in between (11.9 +/- 1.2 microM). The inhibition constants for pyrimethamine increased from 0.19 +/- 0.08 nM (3D7) to 2.0 +/- 0.3 nM (HB3) to 8.9 +/- 0.8 nM (7G8). The inhibition by pyrimethamine of the sensitive clone 3D7 was noncompetitive and competitive for the two other clones. The titration of partially purified DHFR with pyrimethamine revealed a 500-fold increase in the concentration of the drug needed to inhibit the DHFR activity by 50%, when the sensitive clone 3D7 (0.18 +/- 0.02 nM) was compared to the resistant clone 7G8 (95 +/- 16 nM). From the comparison of the specific activities and the catalytic center activities with the Km values for the substrate and the inhibition constants for pyrimethamine, both of which are altered in the resistant clones, we conclude that the molecular mechanism for pyrimethamine resistance in the three clones studied is not based on an overproduction of the DHFR but is due to a decreased affinity to antifolates by a structurally altered enzyme.
Mol Pharmacol 1987 Apr
PMID:Kinetic and molecular properties of the dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant clones of the human malaria parasite Plasmodium falciparum. 355 92

Methanol toxicity is observed in monkeys and humans but is not seen in rats or mice. The expression of methanol poisoning is related to the ability of an animal to metabolize formate to carbon dioxide. Since the rate of formate oxidation is related to hepatic tetrahydrofolate (H4folate) content and the activities of folate-dependent enzymes, studies were designed to determine hepatic concentrations of H4folate and activities of folate-dependent enzymes of human liver and livers of species considered insensitive to methanol poisoning. An excellent correlation between hepatic H4folate and maximal rates of formate oxidation has been observed. In human liver, H4folate levels were only 50% of those observed for rat liver and similar to those found in monkey liver. Total folate was also lower (60% decreased) in human liver than that found in rat or monkey liver. Interestingly, mouse liver contains much higher hepatic H4folate and total folate than rat or monkey liver. This is consistent with higher formate oxidation rates in this species. A second important observation has been made. 10-Formyltetrahydrofolate dehydrogenase activity, the enzyme catalyzing the final step of formate oxidation to carbon dioxide, was markedly reduced in both monkey and human liver. Thus, two mechanisms may be operative in explaining low formate oxidation in species susceptible to methanol toxicity, low hepatic H4folate levels and reduced hepatic 10-formyltetrahydrofolate dehydrogenase activity.
Mol Pharmacol 1987 May
PMID:Studies on the role of folic acid and folate-dependent enzymes in human methanol poisoning. 357 97

The use and metabolism of folates by leishmanias have been studied by assessing the growth of promastigotes in defined media with different folates and the cell content of folate-metabolising enzymes. The folates present in Leishmania mexicana mexicana have been determined using HPLC. Folic acid, 5-formyltetrahydrofolate (THF) and 5-methyl-THF each supported growth of L. m. mexicana promastigotes in defined medium, whereas the parasites did not survive in the absence of folates; p-aminobenzoic acid could not replace the folate requirement. The only folate present at detectable levels in L. m. mexicana promastigotes was 5-methyl-THF. Dihydrofolate reductase (EC 1.5.1.3), methylene-THF reductase (EC 1.1.1.68), serine hydroxymethyltransferase (EC 2.1.2.1) and thymidylate synthetase (EC 2.1.1.45) were all detected in extracts of promastigotes of L. m. mexicana, L. donovani and L. major. Some of these activities were also found in extracts of amastigotes of the former two species. The enzymes of L. m. mexicana have been partially characterised. Methylene-THF reductase may be involved in the conversion in vivo of 5-methyl-THF to 5,10-methylene-THF.
Mol Biochem Parasitol 1987 Mar
PMID:Folate utilisation by Leishmania species and the identification of intracellular derivatives and folate-metabolising enzymes. 357 56

Several fragments of the human dihydrofolate reductase gene (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate NADP+ oxidoreductase, EC 1.5.1.3) were isolated from gene-amplified KB7B cells and characterized. Recombinant plasmids containing intron sequences were constructed. Probes prepared from these plasmids were tested for dihydrofolate reductase precursor mRNA specificity via solution hybridization studies and Northern blot analysis. One probe, p0.69EH, was shown to be specific for dihydrofolate reductase RNA by its greatly enhanced level of hybridization with total RNA from dihydrofolate reductase gene-amplified versus non-amplified cells. In addition, solution hybridization studies with various classes of RNA and Northern blot analysis revealed that p0.69EH hybridizes predominantly with polyadenylated, high molecular weight, nuclear RNA species. Subsequent solution hybridization studies revealed a disproportionate 5-fluorouracil-induced increase in dihydrofolate reductase intron-containing RNA over dihydrofolate reductase mRNA. These results suggest that 5-fluorouracil incorporation into RNA may inhibit the conversion of precursor mRNA to mature mRNA.
Mol Pharmacol 1986 Jun
PMID:5-Fluorouracil augmentation of dihydrofolate reductase gene transcripts containing intervening sequences in methotrexate-resistant KB cells. 371 5


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>