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Kinetics of interaction of HIV reverse transcriptase with primer/template

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Immendörfer,  Ulrike
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Rittinger,  Katrin
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Restle,  Tobias
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;
Molecular chaperones, Max Planck Institute for Medical Research, Max Planck Society;
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

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Goody,  Roger S.
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Divita, G., Mueller, B., Immendörfer, U., Gautel, M., Rittinger, K., Restle, T., et al. (1993). Kinetics of interaction of HIV reverse transcriptase with primer/template. Biochemistry, 32(31), 7966-7971. doi:10.1021/bi00082a018.


Cite as: https://hdl.handle.net/11858/00-001M-0000-0019-AA1C-2
Abstract
Intrinsic protein fluorescence of reverse transcriptases from HIV-1 and HIV-2 provides a sensitive signal for monitoring the interaction of the enzymes with primer/template duplex molecules. Kd values for 18/36-mer DNA/DNA duplexes were found to be in the range of a few nanomolar (about 3 times higher for the enzyme from HIV-2 than for that from HIV-1). The quenching of protein fluorescence induced on binding primer/template, together with an increase in extrinsic fluorescence on interaction with primer/template containing a fluorescent nucleotide at the 3'-end of the primer, was used to investigate the kinetics of interaction with reverse transcriptase from HIV-1. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial association (k(ass) = ca. 5 x 10(8) M-1 s-1) followed by a slow isomerization step (k = ca. 0.5 s-1). These (forward) rate constants are increased in the presence of a non-nucleoside inhibitor (S-TIBO) of HIV-1 reverse transcriptase, while the reverse rate constant for the second step is decreased, leading to an increase in affinity between the enzyme and primer/template by a factor of at least 10 when S-TIBO is bound. The results are discussed in terms of present knowledge of the structure of reverse transcriptase.