STEADY-STATE KINETICS AND INHIBITION OF HIV-1 REVERSE-TRANSCRIPTASE BY A NONNUCLEOSIDE DIPYRIDODIAZEPINONE, BI-RG-587, USING A HETEROPOLYMERIC TEMPLATE

Steady state kinetics and inhibition by a dipyridodiazepinone of the reverse transcriptase from human immunodeficiency virus type 1 (HIV) were studied using a heteropolymeric RNA template with a sequence from the authentic initiation site on the HIV genome. For addition of the first deoxynucleotide to primer, k(cat)/K(M) is 0.05 (nM-min)-1 and K(M) is 10 nM. When all 4 deoxynucleotide triphosphates are present and processive synthesis occurs, catalysis is less efficient; k(cat)/K(M) = .0077 (nM-min)-1 and K(M) = 100 nM for dATP. These results are consistent with a rate determining conformation change involved in translocation of the enzyme along the template. Inhibition by the dipyridodiazepinone BI-RG-587 is noncompetitive with respect to both nucleotide and template-primer; this compound decreases V(max) but does not affect K(M). Thus, this inhibitor binds to a site distinct from the substrate binding sites with K(i) of 220 nM. Inhibition by BI-RG-587 results in a uniform decrease in amount of products of all lengths rather than a shift from longer to shorter products, suggesting the inhibitor does not affect processivity of reverse transcriptase.