Kinetics of interaction of HIV reverse transcriptase with primer/template

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.

Human immunodeficiency virus‐1 reverse transcriptase heterodimer stability

“…A Kd value of < I nM was obtained by Beard and Wilson (1993). Divita et al (1993a) obtained a Kd value of 1.8 nM at 25 "C from a titration curve that measured binding from the observed changes in intrinsic protein fluorescence. It is evident that a variety of experimental approaches yield Kd values for primerkernplate binding that are predominantly in the low nanomolar range.…”

“…From the dissociation rate constant and Kd values, binding rate constants in the range of 106-107 M-I S -I are obtained (Kati et al, 1992;Beard & Wilson, 1993;Hsieh et al, 1993). Stopped-flow kinetics gives a value of 5 x 10' M" s-l (Divita et al, 1993a). Given the slow rate of dissociation of free RT heterodimers to monomers, an estimated tl,* of 53 min at 37 "C, the primer/template binding step is kinetically isolated.…”

Review

“…During these interactions it has been proposed that RT under-goes at least three conformational changes. Divita etal. suggested that HIV-1 RT, in the presence of DNA, changes conformation upon binding to nucleic acids (Divita et al, 1993b). The polymerase is thought to undergo a second conformational change subsequent to dNTP binding immediately prior to the chemical catalysis step.…”

“…HIV-1 RT tightly binds double-stranded nucleic acids with a K^ of 5-38ΠΜ. Structural and biochemical data as well as fluorescence quenching studies suggested that RT binds to DNA by a two step mechanism (Divita et al, 1993b;Kruhoffer et al, 1993). The structure of HIV-1 RT, in the absence of DNA, shows the p66 thumb subdomain folded over the p66 palm subdomain, and coming into contact with the finger subdomain (Raag et al, 1994).…”

Synthesis of 2′,3′,5′-trideoxyuridine-5′-methylphosphonic Acid and its Diphosphate Derivative. Study of the Interaction with HIV-1 reverse Transcriptase

“…HIV-1 RT exhibits a characteristic intrinsic tryptophan fluorescence with a maximal emission at 338 nm due to the 37 tryptophan residues of the enzyme molecule (Divita et al, 1993). The binding of 10 to RT induced a 25% quenching of intrinsic fluorescence.…”