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. 2010 Apr 27;107(17):7734-9.
doi: 10.1073/pnas.0913946107. Epub 2010 Apr 12.

Nucleotide-dependent Conformational Change Governs Specificity and Analog Discrimination by HIV Reverse Transcriptase

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Nucleotide-dependent Conformational Change Governs Specificity and Analog Discrimination by HIV Reverse Transcriptase

Matthew W Kellinger et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Single turnover studies on HIV reverse transcriptase suggest that nucleoside analogs bind more tightly to the enzyme than normal substrates, contrary to rational structural predictions. Here we resolve these controversies by monitoring the kinetics of nucleotide-induced changes in enzyme structure. We show that the specificity constant for incorporation of a normal nucleotide (dCTP) is determined solely by the rate of binding (including isomerization) because isomerization to the closed complex commits the substrate to react. In contrast, a nucleoside analog (3TC-TP, triphosphate form of lamivudine) is incorporated slowly, allowing the conformational change to come to equilibrium and revealing tight nucleotide binding. Our data reconcile previously conflicting reports suggesting that nucleotide analogs bind tighter than normal nucleotides. Rather, dCTP and 3TC-TP bind with nearly equal affinities, but the binding of dCTP never reaches equilibrium. Discrimination against 3TC-TP is based on the slower rate of incorporation due to misalignment of the substrate and/or catalytic residues.

Conflict of interest statement

Conflict of Interest: K.A.J. is President of KinTek Corp., which provided the SF-2004 stopped-flow and RQF-3 quench-flow instruments and KinTek Explorer software.

Figures

Scheme 1.
Scheme 1.
Scheme 2.
Scheme 2.
Fig. 1.
Fig. 1.
Time dependence of fluorescence change during dCTP binding and incorporation. (A) An E·DNA complex was formed using MDCC-labeled HIV RT (0.4 μM) and 25/45(C) DNA oligonucleotide (0.6 μM) and then mixed 1∶1 in the stopped flow with various concentrations of dCTP (to achieve final concentrations of 2, 4, 8, 10, 20, 40, 60, 80, 200 μM dCTP, 200 nM enzyme, and 300 nM DNA). Data from each concentration were fit to a double exponential to derive rates of reaction. (B) Rates of the fast and slow reaction phases (λ1 and λ2) from fitting data in A are graphed as a function of concentration. The slope of the fast phase defines the apparent second-order rate constant for dCTP binding (K1k2 = 7 ± 0.5 μM-1 s-1), whereas the maximum rate of the slow phase defines k3 = 19 ± 0.5 s-1 according to Scheme 2. (C) The experiment shown in A was repeated as lower temperatures (5, 10, 18 °C, lower to upper curves) to estimate the maximum rate of the conformational change, which is too fast to observe at 37 °C. The dashed line corresponds to data collected at 37 °C at sufficiently low concentrations to accurately define the binding rate.
Fig. 2.
Fig. 2.
Global fit to kinetics of dCTP incorporation. In each figure, the smooth lines show the best fit obtained by nonlinear regression in fitting all data simultaneously to the model shown in Scheme 3 using KinTek Explorer software. (A) Data from a chemical-quench-flow experiment in which an E·DNA complex (150 nM enzyme, 100 nM DNA) was mixed with various concentrations of nucleotide (0.25, 0.5, 1, 10, 25 μM), then quenched with 0.5 M EDTA. (B) Fluorescence stopped-flow signal after mixing E·DNA with various nucleotide concentrations (final concentrations: 2, 4, 6, 12, 28, 40, 60, 80, 200 μM dCTP, 200 nM enzyme, 300 nM DNA). (C) Time course of nucleotide dissociation as described in the text; here we only fit the slow phase of the reaction (black line) which occurred at a rate of 0.06 s-1.
Scheme 3.
Scheme 3.
Fig. 3.
Fig. 3.
Global fit to kinetics of 3TC-TP incorporation. (A) Data from a chemical-quench-flow experiment in which an E·DNA complex (150 nM enzyme, 100 nM DNA) was mixed with various concentrations of nucleotide (0.01, 0.02, 0.04, 0.06, 0.1, 1, 2.5, 5, 7.5 μM), then quenched with 0.5 m EDTA. (B) Fluorescence stopped-flow signal after mixing E·DNA (200 nM enzyme, 300 nM DNA) with various nucleotide concentrations (2, 4, 8, 10, 20, 40, 60 μM). For figures A and B, the smooth lines show the best fit obtained by nonlinear regression fitting all data simultaneously to the model shown in Scheme 4 using KinTek Explorer software. (C) Time course of nucleotide dissociation from the E.DNAdd complex was performed as described in the text; fitting the slow phase (black line) required a value of k-4 = 0.6 s-1 (Scheme 4).
Scheme 4.
Scheme 4.
Scheme 5.
Scheme 5.

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