HUMAN-IMMUNODEFICIENCY-VIRUS REVERSE-TRANSCRIPTASE SUBSTRATE-INDUCED CONFORMATIONAL-CHANGES AND THE MECHANISM OF INHIBITION BY NONNUCLEOSIDE INHIBITORS

A combination of transient kinetic and equilibrium titration methods h as been used to show that both primer/template and nucleotide binding to human immunedeficiency virus type 1 (HIV-1) reverse transcriptase a re two-step processes. In both cases, after initial formation of relat ively weakly bound states, isomerization reactions lead to tightly bou nd states, In the case of deoxynucleotide binding to the reverse trans criptase-primer/template complex, the second step in the interaction i s rate-limiting in the overall reaction during processive polymerizati on. Discrimination against incorrect nucleotides occurs both in the in itial weak binding and in the second step but is purely kinetic in the second step (as opposed to thermodynamic in the first step). Nonnucle oside inhibitors have a relatively small effect on nucleotide-binding steps (overall affinity is reduced by a factor of ca. 10), while the a ffinity of the primer/template duplex is increased by at least a facto r of 10, The major effect of nonnucleoside inhibitors is on the chemic al step (nucleotide transfer).