Reverse transcriptases kinetics

Divita G, Rittinger K, Geourjon C, Deleage G, Goody RS. Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase A two step process. J Mol Biol 1994 145 508-521. 

Reverse transcriptase 248, 257, 657 Reversed phase columns 103 Reversible chemical reactions 284 kinetics of 458 Rhabdoviruses 247 Rhamnogalacturonan 177 Rhamnose (Rha) 165s, 180 Rheumatoid arthritis 627 Rhinovirus 247... 

Hsieh, J. C., Zinnen, S., and Modrich, P. (1993). Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase. / Biol. Chem. 268, 24607-24613. 

Wohrl, B. M., Krebs, R., Goody, R. S., and Resde, T. (1999). Refined model for primer/ template binding by HIV-1 reverse transcriptase Pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation events distinguish between different binding modes depending on the nature of the nucleic acid substrate. /. Mol. Biol. 292, 333-344. 

Currens, M. J. Mariner, J. M. McMahon, J. B. Boyd, M. R. Kinetic analysis of inhibition of human immunodeficiency vims type-1 reverse transcriptase by calanolide A. J. Pharmaol. Exp. Then, 1996, 279 652 61. 

Yao, E.M., Tavis, J.E. Kinetics of synthesis and turnover of the duck hepatitis B virus reverse transcriptase. J. Biol. Chem. 2003 278 1201-1205... 

Analyses of in situ DNA synthesis of Euglena gracilis identify zinc-dependent steps in the eukaryotic cell cycle and show that the derangements in RNA metabolism are critical determinants of the growth arrest associated with zinc deficiency. Combined use of microwave-induced emission spectrometry and micro gel emulsion chromatography shows the presence of stoichiometric amounts of zinc essential to the function of E. gracilis and yeast RNA polymerases, the reverse transcriptases" from avian myeloblastosis, murine leukemic and woolly type C viruses, and E. coli methionyl tRNA synthetase. These results stress the importance of zinc to both nucleic acid and protein metabolism. Transient-state kinetic studies of carboxypeptidase A show that zinc functions in the catalytic step of peptide hydrolysis and in the binding step of ester hydrolysis. 

Figure 10.16 Steady-state kinetic efficiencies for single-nucleotide insertion by HIV-1 reverse transcriptase to duplex RNA templates [5 -r(ACU GXU CUC CCU AUA GUG AGU CGU AUU A),-55 -d(T AAT ACG ACT CAC TAT AGG GAG A)] with dATP.

Zhang, H Domadula, G., Wu, Y., Havlir, D., Richman, D. D., and Pomerantz, R. J. (1996) Kinetic analysis of intravirion reverse transcription in the blood plasma of human immunodeficiency virus type 1-infected individuals direct assessment of resistance to reverse transcriptase inhibitors in vivo../. Virol. 70,628-634. 

Balzarini, J., Perez-Perez, M.-J., San-F61ix, A., Camarasa, M.-J., Bathurst, I. C., Barr, P. J., and De Clercq, E. (1992) Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue l-[2, 5 -bis-0-(tert-butyldimethylsilyl)-p-D-ribofuranosyl]-3,-spiro-5"-[4"-amino-l",2"-oxathiole-2",2"-dioxide]-thymine (TSAO-T)../. Biol. Chem. 267, 11,831-11,838. 

Althaus, I., J. Chou, A. Gonzales, M. Deibel, K. Chou, F. Kezdy, D. Romero, J. Palmer, R. Thomas, P. Aristoff, W. Tarpley, and F. Reusser. Kinetic Studies with the Non-Nucleoside HlV-1 Reverse Transcriptase Inhibitor U-88204E. Biochemistry 6548-6554 (1993). [How enzyme kinetics can play a role in AIDS research.]... 

In this book I have tried to give as many life science-related examples as possible and although the general concepts of the problems are correct, often the parameters of reactions are fictional. For example, it is well known that HTV reverse transcriptase forms a dimer however the rate constant of this process, as used in chapter 5, has been set more or less arbitrarily. This book is therefore NOT intended as a reference for accurate numbers, but should rather exemplify concepts of reaction kinetics in a Bioscience-context. 

The protein reverse transcriptase (RT) is a viral protein essential for the propagation of retroviruses like HIV. RT is initially produced in the cell as a monomer , but quickly forms a homo-dimer , which is a complex of two RT-monomers. Only this homo-dimer is active, whereas RT monomers are not. Obviously there is a huge interest to find a potent drug against HIV and this can be done by investigating the kinetics of RT dimer formation. If one could stop the formation of RT-dimers then ftie virus could no longer replicate. To study the formation of RT dimers you use a concentration of 200 nM RT monomers and measure their concentration at certain time intervals ... 

A further important step in quantitative RT-PCR is the production of a single-stranded (ss) complementary DNA copy (cDNA) of the RNA through the reverse transcriptase (RT). Its dynamic range, sensitivity, and specificity are of prime consideration for a successful kinetic RT-PCR assay. For many quantitative applications, moloney murine leukaemia virus (MMLV) RT is the enzyme of choice, as its cDNA synthesis rate is up to 50-fold greater than that of avian myeloblastosis virus (AMV). Newly available thermostable enzymes maintain their activity up to 70°C, thus permitting increased specificity and efficiency of first primer annealing. Fiowever, this enzyme type may be less robust than more conventional ones as it appears to be more sensitive to inhibitors present in RNA preparation. 

Gudima SO, Memelova LV, Borodulin VB, Pokholok DK, Mednikov BM, Tolkachev ON, Kochetkov SN (1994) Kinetic analysis of interaction of human immunodeficiency virus reverse transcriptase with alkaloids. Mol Biol 28 1308-1314... 

Reverse transcriptase (RT) plays a critical role in the early steps of the life of human immunodeficiency virus (HIV) (304), and for over a decade has been one of the major targets of AIDS therapy. Polycitone A (280) was found to be a potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases (305). Polycitone A exhibited potent inhibitory capacity of both RNA- and DNA-directed DNA polymerases. It inhibits retroviral reverse transcriptases (RTs) of human immunodeficiency virus type 1 (HIV), murine leukemia virus (MLV) and mouse mammary tumor virus (MMTV)] as efficiently as cellular DNA polymerases of both DNA polymerases a and p and the prokaryotic Klenow fragment of Escherichia coli DNA polymerase I. The mode and mechanism of inhibition of the DNA-polymerase activity associated with HIV-1 RT by polycitone A (280) have been studied. The results suggest that the inhibitory capacity of the DNA polymerase activity is independent of the template-primer used. The RNase H function is hardly affected by this inhibitor. Polycitone A has been shown to interfere with DNA primer extension, as well as with the formation of the RT-DNA complex. Steady-state kinetic studies demonstrate that this inhibitor can be considered as an allosteric inhibitor of HIV-1 RT. The target site on the enzyme may be also spatially related to the... 

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