HIV-1 reverse transcriptase resistance to nonnucleoside inhibitors

The parameters governing the polymerization mechanism of reverse transcriptase containing the tyrosine to cysteine mutation at position 181 (Y181C) were determined using pre-steady-state techniques. The pathway for single nucleotide incorporation catalyzed by Y181C is similar to that determined for wild-type RT where a rate-limiting conformational change precedes fast chemistry and is followed by slow steady-state release of the primer/template. The Y181C mutant enzyme binds a 25/45-mer duplex DNA tightly with a K-d of 11 nM. However, the Y181C mutation weakens the nucleotide affinity 2-3-fold relative to the wild-type complex. We also determined the parameters governing the mechanism of nonnucleoside inhibitor resistance with Y181C. The K-d value of Nevirapine with the mutant E . DNA complex increased approximately 500-fold. The decreased affinity of Nevirapine for the mutant enzyme is a consequence of a faster inhibitor dissociation rate from the enzyme complex of Y181C relative to that of the wild-type. The E . DNA complex of Y181C may be saturated with Nevirapine, and the I . E . DNA complex is capable of a maximum incorporation rate of 0.1 s(-1) (a 10-fold faster rate than that of the wild-type I . E . DNA complex). The overall two-step binding of nucleotide to Y181C in the presence of Nevirapine remains unaffected.