1 research outputs found
Thermodynamics of HIV‑1 Reverse Transcriptase in Action Elucidates the Mechanism of Action of Non-Nucleoside Inhibitors
HIV-1 reverse transcriptase (RT)
is a heterodimeric enzyme that
converts the genomic viral RNA into proviral DNA. Despite intensive
biochemical and structural studies, direct thermodynamic data regarding
RT interactions with its substrates are still lacking. Here we addressed
the mechanism of action of RT and of non-nucleoside RT inhibitors
(NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC
approach, a step-by-step thermodynamic dissection of the RT polymerization
activity showed that most of the driving force for DNA synthesis is
provided by initial dNTP binding. Surprisingly, thermodynamic and
kinetic data led to a reinterpretation of the mechanism of inhibition
of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered
by a kinetic barrier and NNRTIs mostly interact with free RT. Once
formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent
orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA
complexes being unable to bind the incoming nucleotide substrate