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Biochemistry. 1996 Jan 23;35(3):1054-63.

HIV-1 reverse transcriptase resistance to nonnucleoside inhibitors.

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Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.


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 Kd 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 Kd 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.

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