Steady state kinetics and inhibition of HIV-1 reverse transcriptase by a non-nucleoside dipyridodiazepinone, BI-RG-587, using a heteropolymeric template.

Abstract:

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, kcat/KM is 0.05 (nM-min)-1 and KM is 10 nM. When all 4 deoxynucleotide triphosphates are present and processive synthesis occurs, catalysis is less efficient; kcat/KM = .0077 (nM-min)-1 and KM = 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 Vmax but does not affect KM. Thus, this inhibitor binds to a site distinct from the substrate binding sites with Ki 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.

Polymerases:

Topics:

Kinetic Parameters

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
HIV RT Kopp EB1991 KM 10nM Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State
HIV RT Kopp EB1991 KM 40nM Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Steady State
HIV RT Kopp EB1991 KM 20nM Reaction: Nucleotide incorporation; Substrate: dGTP; Technique: Steady State
HIV RT Kopp EB1991 KM 100nM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Steady State
HIV RT Kopp EB1991 kcat 0.5 /minute Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State
HIV RT Kopp EB1991 kcat 0.43 /minute Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Steady State
HIV RT Kopp EB1991 kcat 0.12 /minute Reaction: Nucleotide incorporation; Substrate: dGTP; Technique: Steady State
HIV RT Kopp EB1991 kcat 0.77 /minute Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Steady State

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