KINETICS OF INTERACTION OF HIV REVERSE-TRANSCRIPTASE WITH PRIMER TEMPLATE

Intrinsic protein fluorescence of reverse transcriptases from HIV-1 an d HIV-2 provides a sensitive signal for monitoring the interaction of the enzymes with primer/template duplex molecules. K(d) values for 18/ 36-mer DNA/DNA duplexes were found to be in the range of a few nanomol ar (about 3 times higher for the enzyme from HIV-2 than for that from HIV-1). The quenching of protein fluorescence induced on binding prime r/template, together with an increase in extrinsic fluorescence on int eraction with primer/template containing a fluorescent nucleotide at t he 3'-end of the primer, was used to investigate the kinetics of inter action with reverse transcriptase from HIV-1. The results can be expla ined in terms of a two-step binding model, with a rapid diffusion-limi ted 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 const ants are increased in the presence of a non-nucleoside inhibitor (S-TI BO) of HIV-1 reverse transcriptase, while the reverse rate constant fo r the second step is decreased, leading to an increase in affinity bet ween 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.