Abstract
Reverse transcriptase (RNA-directed DNA polymerase, EC 2.7.7.49) of human immunodeficiency virus type 1 has been examined with respect to the steady-state kinetics of polymerization of dNTPs into product DNA. With dNTPs as variable substrate, the kinetics of polymerization deviated from standard Michaelis-Menten kinetics. Substrate inhibition was observed at high substrate concentrations and negative cooperativity was seen at lower substrate concentrations. Examination of incorporation of substrate dNMPs in the presence of nucleotides not complementing the template demonstrated that dNTPs may act as noncompetitive inhibitors, as well as substrate. The Ki of the enzyme for dNTPs was 104 microM. A working model is presented that accounts for the substrate inhibition. In this model, the reverse transcriptase is a multisubunit holoenzyme, where noncompetitive inhibition is mediated by one subunit binding nucleotide and down-regulating the enzymatically active 64-kDa subunit. With additional assumptions, this model can accommodate the negative cooperativity observed.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alizon M., Wain-Hobson S., Montagnier L., Sonigo P. Genetic variability of the AIDS virus: nucleotide sequence analysis of two isolates from African patients. Cell. 1986 Jul 4;46(1):63–74. doi: 10.1016/0092-8674(86)90860-3. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Coffin J. M. Genetic variation in AIDS viruses. Cell. 1986 Jul 4;46(1):1–4. doi: 10.1016/0092-8674(86)90851-2. [DOI] [PubMed] [Google Scholar]
- Gyorkey F., Melnick J. L., Sinkovics J. G., Gyorkey P. Retrovirus resembling HTLV in macrophages of patients with AIDS. Lancet. 1985 Jan 12;1(8420):106–106. doi: 10.1016/s0140-6736(85)91995-6. [DOI] [PubMed] [Google Scholar]
- Haase A. T. Pathogenesis of lentivirus infections. Nature. 1986 Jul 10;322(6075):130–136. doi: 10.1038/322130a0. [DOI] [PubMed] [Google Scholar]
- Hansen J., Schulze T., Mellert W., Moelling K. Identification and characterization of HIV-specific RNase H by monoclonal antibody. EMBO J. 1988 Jan;7(1):239–243. doi: 10.1002/j.1460-2075.1988.tb02805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hansen J., Schulze T., Moelling K. RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. J Biol Chem. 1987 Sep 15;262(26):12393–12396. [PubMed] [Google Scholar]
- Huber H. E., McCoy J. M., Seehra J. S., Richardson C. C. Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching. J Biol Chem. 1989 Mar 15;264(8):4669–4678. [PubMed] [Google Scholar]
- Kedar P. S., Abbotts J., Kovács T., Lesiak K., Torrence P., Wilson S. H. Mechanism of HIV reverse transcriptase: enzyme-primer interaction as revealed through studies of a dNTP analogue, 3'-azido-dTTP. Biochemistry. 1990 Apr 17;29(15):3603–3611. doi: 10.1021/bi00467a003. [DOI] [PubMed] [Google Scholar]
- Klotz I. M., Langerman N. R., Darnall D. W. Quaternary structure of proteins. Annu Rev Biochem. 1970;39:25–62. doi: 10.1146/annurev.bi.39.070170.000325. [DOI] [PubMed] [Google Scholar]
- Koshland D. E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966 Jan;5(1):365–385. doi: 10.1021/bi00865a047. [DOI] [PubMed] [Google Scholar]
- Larder B. A., Darby G., Richman D. D. HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science. 1989 Mar 31;243(4899):1731–1734. doi: 10.1126/science.2467383. [DOI] [PubMed] [Google Scholar]
- Larder B. A., Kemp S. D., Purifoy D. J. Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4803–4807. doi: 10.1073/pnas.86.13.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leeds J. M., Slabaugh M. B., Mathews C. K. DNA precursor pools and ribonucleotide reductase activity: distribution between the nucleus and cytoplasm of mammalian cells. Mol Cell Biol. 1985 Dec;5(12):3443–3450. doi: 10.1128/mcb.5.12.3443. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Levitzki A., Koshland D. E., Jr The role of negative cooperativity and half-of-the-sites reactivity in enzyme regulation. Curr Top Cell Regul. 1976;10:1–40. doi: 10.1016/b978-0-12-152810-2.50008-5. [DOI] [PubMed] [Google Scholar]
- Lightfoote M. M., Coligan J. E., Folks T. M., Fauci A. S., Martin M. A., Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol. 1986 Nov;60(2):771–775. doi: 10.1128/jvi.60.2.771-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Majumdar C., Abbotts J., Broder S., Wilson S. H. Studies on the mechanism of human immunodeficiency virus reverse transcriptase. Steady-state kinetics, processivity, and polynucleotide inhibition. J Biol Chem. 1988 Oct 25;263(30):15657–15665. [PubMed] [Google Scholar]
- Mitsuya H., Jarrett R. F., Matsukura M., Di Marzo Veronese F., DeVico A. L., Sarngadharan M. G., Johns D. G., Reitz M. S., Broder S. Long-term inhibition of human T-lymphotropic virus type III/lymphadenopathy-associated virus (human immunodeficiency virus) DNA synthesis and RNA expression in T cells protected by 2',3'-dideoxynucleosides in vitro. Proc Natl Acad Sci U S A. 1987 Apr;84(7):2033–2037. doi: 10.1073/pnas.84.7.2033. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mitsuya H., Weinhold K. J., Furman P. A., St Clair M. H., Lehrman S. N., Gallo R. C., Bolognesi D., Barry D. W., Broder S. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc Natl Acad Sci U S A. 1985 Oct;82(20):7096–7100. doi: 10.1073/pnas.82.20.7096. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Müller B., Restle T., Weiss S., Gautel M., Sczakiel G., Goody R. S. Co-expression of the subunits of the heterodimer of HIV-1 reverse transcriptase in Escherichia coli. J Biol Chem. 1989 Aug 25;264(24):13975–13978. [PubMed] [Google Scholar]
- Perutz M. F. Regulation of oxygen affinity of hemoglobin: influence of structure of the globin on the heme iron. Annu Rev Biochem. 1979;48:327–386. doi: 10.1146/annurev.bi.48.070179.001551. [DOI] [PubMed] [Google Scholar]
- Poch O., Sauvaget I., Delarue M., Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989 Dec 1;8(12):3867–3874. doi: 10.1002/j.1460-2075.1989.tb08565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ratner L., Fisher A., Jagodzinski L. L., Mitsuya H., Liou R. S., Gallo R. C., Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57–69. doi: 10.1089/aid.1987.3.57. [DOI] [PubMed] [Google Scholar]
- Reardon J. E., Miller W. H. Human immunodeficiency virus reverse transcriptase. Substrate and inhibitor kinetics with thymidine 5'-triphosphate and 3'-azido-3'-deoxythymidine 5'-triphosphate. J Biol Chem. 1990 Nov 25;265(33):20302–20307. [PubMed] [Google Scholar]
- Restle T., Müller B., Goody R. S. Dimerization of human immunodeficiency virus type 1 reverse transcriptase. A target for chemotherapeutic intervention. J Biol Chem. 1990 Jun 5;265(16):8986–8988. [PubMed] [Google Scholar]
- Ricchetti M., Buc H. Reverse transcriptases and genomic variability: the accuracy of DNA replication is enzyme specific and sequence dependent. EMBO J. 1990 May;9(5):1583–1593. doi: 10.1002/j.1460-2075.1990.tb08278.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Skoog L., Bjursell G. Nuclear and cytoplasmic pools of deoxyribonucleoside triphosphates in Chinese hamster ovary cells. J Biol Chem. 1974 Oct 25;249(20):6434–6438. [PubMed] [Google Scholar]
- Weber J., Grosse F. Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA. Nucleic Acids Res. 1989 Feb 25;17(4):1379–1393. doi: 10.1093/nar/17.4.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
- di Marzo Veronese F., Copeland T. D., DeVico A. L., Rahman R., Oroszlan S., Gallo R. C., Sarngadharan M. G. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science. 1986 Mar 14;231(4743):1289–1291. doi: 10.1126/science.2418504. [DOI] [PubMed] [Google Scholar]