Kinetics of interaction of HIV reverse transcriptase with primer/template.
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.
Divita
G
G
Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Müller
B
B
Immendörfer
U
U
Gautel
M
M
Rittinger
K
K
Restle
T
T
Goody
R S
RS
D001483:Base Sequence; D001569:Benzodiazepines / Q000494:pharmacology; D004277:DNA, Single-Stranded / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic; D013871:Thiones / Q000494:pharmacology
D004277:DNA, Single-Stranded; D007093:Imidazoles; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D013871:Thiones; D001569:Benzodiazepines; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase