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)

Nalidixic acid, a very specific inhibitor of bacterial DNA synthesis, has been studied for its action on the avian myeloblastosis virus reverse transcriptase activity. The drug inhibited the DNA synthesis reaction catalyzed by the viral enzyme in the presence of different template-primers. The inhibitory effect by nalidixic acid was higher with polyriboadenylic acid than with polyribocytidylic acid as a synthetic template. With activated DNA as a template nalidixic acid preferentially inhibited the TMP incorporation when compared with the dAMP incorporation. Both these results showed the importance of the presence of adenine in the templates for a more efficient inhibition by nalidixic acid. The inhibition for this drug was also shown in the presence of Mn2+ instead of Mg2+ as the divalent cation, and with a 2'-fluorinated analogue of polyriboadenylic acid as the template. Kinetic data showed a non-competitive inhibition by nalidixic acid in relation to polyriboadenylic acid and to TTP in the reaction catalyzed by reverse transcriptase.
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PMID:Avian myeloblastosis virus reverse transcriptase inhibition by nalidixic acid. 172 88

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

Using affinity purified human immunodeficiency virus (HIV) reverse transcriptase the reaction assay conditions were determined. The optimum incorporation of dTMP into the (rA)n(dT)10 template with HIV reverse transcriptase required 6 mM MgCl2 and 80 mM KCl. The template specificity of HIV reverse transcriptase is quite different from those of the human gamma-polymerase-associated reverse transcriptase or avian virus reverse transcriptase. The preferential inhibition of HIV reverse transcriptase as compared to human gamma-reverse transcriptase was observed with several nucleoside analog triphosphates. The Ki values for thymidine triphosphate analogs with HIV reverse transcriptase ranged from 5 to 13 nM with decreasing effectiveness for 3'-fluoro greater than 3'-amino greater than 2',3'-dideoxy greater than 3'-azido groups. This study provides information on the structure activity relationships of the triphosphate analogs inhibitory effects on HIV reverse transcriptase versus human gamma-polymerase-associated reverse transcriptase, and the possible mechanisms of action of 3' azido thymidine and the 2',3'-dideoxynucleosides, and also identifies other nucleoside analogs for possible development as inhibitors of HIV.
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PMID:Human immunodeficiency virus reverse transcriptase. General properties and its interactions with nucleoside triphosphate analogs. 243 77

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

The fidelity of DNA synthesis by reverse transcriptases from human immunodeficiency virus and other retroviruses was compared by measuring the rates of misincorporation of dCMP in the place of TMP in cell-free DNA synthesis with polyadenylic acid as the template. The fidelity of human immunodeficiency virus reverse transcriptase was found to be about one-third of that of the reverse transcriptases of other retroviruses.
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PMID:Low fidelity of cell-free DNA synthesis by reverse transcriptase of human immunodeficiency virus. 245 89

A study of steady-state kinetics of polymerization by purified human immunodeficiency virus DNA polymerase (reverse transcriptase) has been conducted. DNA synthesis was examined using a system of poly(rA) as template, oligo(dT) as primer, and dTTP as nucleotide substrate. The substrate initial velocity patterns point to an ordered mechanism with template-primer adding first. Product inhibition kinetics with either pyrophosphate or phosphonoformate are consistent with this mechanism. The human immunodeficiency virus reverse transcriptase acts processively in this replication system, but exhibits some probability of terminating after each dTMP addition to the nascent chain. The probability of terminating was approximately 20-fold higher after the first dTMP addition than after subsequent additions. With this information on the mode of polymerization, appropriate kinetic models and steady-state rate equations are discussed. In further studies, we found that a heterologous polynucleotide, poly(rC), is a potent inhibitor of the enzyme. The pattern of this inhibition is uncompetitive against template-primer, suggesting that interaction with free enzyme is not the mechanism of the inhibition.
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PMID:Studies on the mechanism of human immunodeficiency virus reverse transcriptase. Steady-state kinetics, processivity, and polynucleotide inhibition. 245 25

Reverse transcriptase from the human immunodeficiency virus type I (HIV-1) was expressed in E. coli and purified to near homogeneity. The enzyme was shown to contain reverse transcriptase, DNA polymerase and ribonuclease H activities. The DNA polymerase activity converted singly-primed phi X174 (+) DNA into the double-stranded form. Two third of the replication product is ligatable to covalently closed circular DNA (RFIV-form DNA) indicating that DNA synthesis by HIV reverse transcriptase can proceed until the enzyme matches the 5'-end of a pre-existing primer molecule. The in vitro accuracy of HIV reverse transcriptase was measured with the phi X174am16 reversion assay to be 1/7,400. Reversion rates for the individual mispairs were determined from pool bias studies to be 1/8,000 for the dGMP:T template mismatch, 1/35,000 for the dGMP:A template mismatch, 1/45,000 for the dAMP:G template mismatch, 1/73,000 for the dCMP:T template mispair, 1/140,000 for the dCMP:A template mispair, and 1/180,000 for the dGMP:G template mismatch. The dTMP:T template mispair was below the detection limit of the assay indicating a reversion rate of less than 1/300,000 for this particular mispair.
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PMID:Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA. 246 38

The 2',3'-dideoxynucleoside triphosphates (ddNTPs) are potent substrate analog inhibitors of human immunodeficiency virus (HIV) reverse transcriptase and have clinical utility in the treatment of acquired immunodeficiency syndrome. Several issues regarding the interaction of these compounds with HIV reverse transcriptase were examined. The potency of unsubstituted ddNTPs and the 3'-azido analog of dTTP (AZTTP) was influenced by the choice of template. Both compounds were more potent with the complementary homopolymer templates than with gapped duplex DNA, although the Km for the competing dNTP was similar with different templates. The Ki for AZTTP was greater than for the unsubstituted ddNTPs with either a homopolymer or a gapped duplex DNA template. HIV reverse transcriptase incorporated ddCMP and AZTMP into primed phage m13 DNA at sites specified for insertion of dCMP and dTMP, respectively. ddCTP was more efficiently utilized as a substrate than was AZTTP. Primer elongation due to base misincorporation was observed in the absence of one dNTP. The combined effect of ddNTPs and the pyrophosphate analog phosphonoformate (PFA) on HIV reverse transcriptase was also examined, and inhibition by PFA in combination with ddTTP or AZTTP was mutually exclusive.
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PMID:Inhibition of human immunodeficiency virus reverse transcriptase by 2',3'-dideoxynucleoside triphosphates: template dependence, and combination with phosphonoformate. 247 97

We have investigated the ability of some nucleoside 5'-triphosphate analogues to terminate the DNA synthesis catalyzed by calf thymus DNA polymerase alpha and terminal deoxynucleotidyl transferase, rat liver DNA polymerase beta, E. coli DNA polymerase I (Klenow's fragment) and AMV reverse transcriptase. It has been shown that lyxoanhydronucleoside 5'-triphosphates terminate DNA synthesis catalyzed by reverse transcriptase and terminal deoxynucleotydil transferase. 2',3'-O-Isopropylidenecytidine 5'-triphosphate inhibits the DNA synthesis catalyzed by reverse transcriptase and DNA polymerase beta and its moiety was incorporated in the place of dTMP residue. Riboanhydroadenosine 5'-triphosphate reveals the properties of an effective termination substrate for all the DNA polymerases studied. This is the first attempt to investigate nucleotide analogues with the restricted conformation of the carbohydrate moiety as termination substrates for several prokaryotic and eukaryotic DNA polymerases.
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PMID:[Conformation limited nucleoside-5'-phosphates as termination substrates for DNA-polymerases]. 248 45

The characteristics of the virion-associated RNA-dependent DNA polymerase (RDPase) of a baboon endogenous virus, M7, were studied extensively; the optimal conditions for the exogenous RDPase reaction were obtained with 0.4 mM-Mn2+, 110 mM-NaCl, 24 microgram/ml poly(rA). oligo(dT)12-18, at pH 7.6 in the presence of 0.01 % Brij-58. Under these conditions, the incorporation of 3H-TMP proceeded up to 90 min at a speed of 0.1 pmol TMP/microgram virus protein/min. Poly(rC). oligo (dG)12-18 and poly(rCm). oligo (dG)12-18 served as the template-primers in the exogenous reaction with 3 mM-Mg2+ and 0.4 mM-Mn2+, respectively. Polyuridylic acid, bleomycin, rifampicin, spermidine and inorganic phosphate significantly inhibited the RDPase activity of BaEV. The RDPase of BaEV requires a higher concentration of NaCl, a lower pH and milder conditions of detergent treatment than those of other mammalian retroviruses.
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PMID:The characteristics of the RNA-dependent DNA polymerase of baboon endogenous virus. 615 24


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