Nucleotide-dependent conformational change governs specificity and analog discrimination by HIV reverse transcriptase

Proc Natl Acad Sci U S A. 2010 Apr 27;107(17):7734-9. doi: 10.1073/pnas.0913946107. Epub 2010 Apr 12.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Affinity
  • Chromatography, Ion Exchange
  • Cytidine Triphosphate / analogs & derivatives*
  • Cytidine Triphosphate / metabolism
  • DNA Primers / genetics
  • Dideoxynucleotides / metabolism*
  • Fluorometry
  • HIV Reverse Transcriptase / genetics*
  • HIV Reverse Transcriptase / metabolism*
  • Kinetics
  • Lamivudine / analogs & derivatives*
  • Lamivudine / metabolism
  • Models, Chemical
  • Mutagenesis
  • Protein Binding / genetics
  • Protein Conformation*
  • Substrate Specificity / genetics


  • DNA Primers
  • Dideoxynucleotides
  • lamivudine triphosphate
  • Lamivudine
  • Cytidine Triphosphate
  • HIV Reverse Transcriptase