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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Captan was used as an inhibitor of avian myeloblastosis virus reverse transcriptase to study the polymerase and RNase H catalytic activities. With purified enzyme, RNase H activity was 10-fold more sensitive to captan than was either the DNA-dependent or RNA-dependent DNA polymerase activity. Inhibition of the RNA-dependent polymerase activity could be prevented by dTTP. Conversely, inhibition of this polymerase activity was enhanced by template/primer. The calculated KdTTP of the uninhibited reaction was 5.6 microM. Kinetic studies allow for the proposition of a model for the interaction of captan with the polymerase active center. RNase H activity showed a sigmoidal relationship between activity and substrate concentration. Nuclease activity decreased in Vmax with no change in the Hill coefficient in the presence of captan. Addition of dithiothreitol to the incubation cocktail prevented inhibition by captan of both RNA-dependent polymerase and RNase H activities, suggesting that the (trichloromethyl)thio moiety of captan is involved in the inhibitory action. Captan inhibition suggests the presence of essential amino residues in both polymerase and RNase H active centers.
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PMID:Differential effects of captan on DNA polymerase and ribonuclease H activities of avian myeloblastosis virus reverse transcriptase. 242 94

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. & Broder, S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentrations above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5'-mono-, di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di- and triphosphate were low (less than or equal to 5 microM and less than or equal to 2 microM, respectively). Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent Km values of 2.9 microM and 3.0 microM. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 microM vs. 4.1 microM), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. IC50 (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase alpha. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase alpha. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.
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PMID:Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. 243 Feb 86

Inhibitory effects of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) and its three derivatives modified on the 3' position of ribose moiety [3'-azido-2',3'-dideoxythymidine 5'-triphosphate (3'-N3-ddTTP), 3'-amino-2',3'-dideoxythymidine 5'-triphosphate (3'-NH2-ddTTP) and 2'-deoxyxylo-furanosylthymine 5'-triphosphate (dXTP)] on the activity of the reverse transcriptase purified from Rauscher murine leukemia virus were examined and compared with each other. When (rA)n X (dT)12-18 was used as the template X primer in the presence of manganese ion, all these compounds except 3'-NH2-ddTTP inhibited the reverse transcriptase activity in competitive fashion with respect to the dTTP substrate. The inhibition potentials of these compounds are ordered as follows: 3'-N3-ddTTP (Ki = 1.8 microM) greater than ddTTP (Ki = 9.3 microM) greater than dXTP (Ki = 16.3 microM), and the Ki values of these inhibitors are smaller than the Km of dTTP (30 microM). The observed inhibitions were mainly due to competition between the dTTP substrate and inhibitor rather than chain-termination of the elongating DNAs caused by incorporation of these dideoxy compounds.
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PMID:Inhibition of reverse transcriptase activity by 2',3'-dideoxythymidine 5'-triphosphate and its derivatives modified on the 3' position. 243 May 67

Human T cell lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus is the etiologic agent of the acquired immune deficiency syndrome (AIDS) and AIDS-related complex. The effect of 3'-azido-3'-deoxythymidine (AZT) on the HTLV-III/lymphadenopathy-associated virus infection was quantitatively studied with HTLV type I-carrying MT-4 cells. The AZT compound inhibited HTLV-III-induced cytopathic effect and virus-specific antigen expression in MT-4 cells at concentrations of 5 and 10 microM. In addition, a plaque-forming assay was performed to assess the effect of AZT on virus replication in MT-4 cells freshly infected with HTLV-III and in continuous HTLV-III-producing Molt-4/HTLV-III cells. Results showed that AZT efficiently and effectively inhibited the replication of HTLV-III in infected MT-4 cells. AZT is a strong inhibitor of reverse transcriptase activity of HTLV-III as a triphosphate, to such a degree that even 1.0 pM azido-TTP inhibits 50% of reverse transcriptase activity. However, it did not show any effect in the HTLV-III-producing cell line Molt-4/HTLV-III. Thus, AZT has no effect on virus replication of an already integrated virus. When 5 microM AZT was added to HTLV-III-infected MT-4 within 20 h after infection, a striking suppressive effect was noticed. This concentration was much lower than that which inhibits the growth of MT-4 cells. These results confirm those found in a previous report (H. Mitsuya, K. J. Weinhold, P. S. Furman, H. S. Clair, S. N. Lehrman, R. C. Gallo, D. Bolognesi, D. W. Barry, and S. Broder, Proc. Natl. Acad. Sci. USA 82:7096-7100, 1985) and suggest that AZT might be used as an experimental antiviral agent for AIDS and AIDS-related complex.
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PMID:Inhibition of replication and cytopathic effect of human T cell lymphotropic virus type III/lymphadenopathy-associated virus by 3'-azido-3'-deoxythymidine in vitro. 243 24

Viral reverse transcriptase activity was inhibited in a concentration dependent manner by 2',5'-oligoadenylate. Kinetically this inhibition was of a mixed type where 2',5'-oligoadenylate was not strictly competitive with dTTP. The potency of inhibition was more marked in the absence than in the presence of sulfhydryl agents. 2',5'-oligoadenylate had no effect on DNA-dependent E. coli DNA polymerase and was much less active against mammalian DNA polymerases. This is the first report of reverse transcriptase inhibition by an inducible constitutive natural ligand.
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PMID:Inhibition of viral reverse transcriptase by 2',5'-oligoadenylates. 243 79

The two isomers 3'-azido-3'-deoxythymidine 5'-triphosphate (erythro-AZT-TP) and 1-(3'-azido-2',3'-dideoxy-beta-D-xylofuranosyl)thymine 5'-triphosphate (threo-AZT-TP) were studied as inhibitors of the reverse transcriptase activity of avian myelobastosis virus. Kinetic analysis of the (rA)n X (dT)12-18 (a standard template primer complex of polyriboadenylate and oligodeoxythymidylate of indicated length)-directed reaction revealed that erythro-AZT-TP was a competitive inhibitor with respect to dTTP, whereas threo-AZT-TP was a noncompetitive inhibitor. The apparent Ki values, as calculated from Dixon plots, were 0.48 and 5.5 microM, respectively, compared with a Km value for dTTP of about 70 microM. These results indicate that erythro-AZT-TP had an approximately 150-times-higher affinity to the enzyme than dTTP had and that the avian myeloblastosis virus reverse transcriptase had different binding sites for the two isomers.
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PMID:Different patterns of inhibition of avian myeloblastosis virus reverse transcriptase activity by 3'-azido-3'-deoxythymidine 5'-triphosphate and its threo isomer. 244 Mar 79

3'-Azido-3'-deoxythymidine triphosphate (erythro) and its threo isomer were synthesized and investigated for their inhibition of HIV reverse transcriptase from virus isolate U 937/HTLV-III. The erythro isomer was a competitive inhibitor of the reaction directed by (rA)n(dT)12-18 and the Ki value was 0.0022 microM. The threo isomer was at least 100-fold less active. The inhibition was specific for dTMP incorporation, and dGMP incorporation using (rC)n(dG)12-18 as template/primer was not affected. The Km value for dTTP varied between 0.7 microM and 1.7 microM. Reverse transcriptase from nine HIV isolates were tested for inhibition by the erythro isomer and only slight differences in sensitivity were observed.
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PMID:Inhibition of the reverse transcriptase from HIV by 3'-azido-3'-deoxythymidine triphosphate and its threo analogue. 244 Mar 80

3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.
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PMID:DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis. 244 69

The sugar-modified dTTP analogues 2',3'-didehydro-2',3'-dideoxy-thymidine 5'-triphosphate (ddeTTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), 3'-fluorothymidine 5'-triphosphate (FdTTP), and 3'-azidothymidine 5'-triphosphate (N3dTTP) are demonstrated to be very effective and selective inhibitors of the HIV-associated reverse transcriptase (HIV-RT). This conclusion is based on a comparison of the ID50 values of the compounds for the HIV-RT (ranging from 0.03 microM for ddeTTP to 0.1 microM for ddTTP) and the cellular DNA polymerase alpha (greater than 200 microM). DNA polymerase beta is partially affected by N3dTTP (ID50 = 31 microM) and by the other analogues (ID50 = 1-2.2 microM). FdTTP has proved as effective as N3dTTP (ID50 = 0.05 microM) in suppressing the HIV-RT activity. Kinetic analysis revealed for both dTTP analogues a competitive type of inhibition and the same K1 values (about 0.05 microM).
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PMID:Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5'-triphosphate in comparison to cellular DNA polymerases alpha and beta. 244 44

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional amino-terminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptase-related protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies.
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PMID:Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. 244 94


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