Rate-Limiting Pyrophosphate Release by HIV Reverse Transcriptase and its Effect on Enzyme Specificity

Abstract

HIV-1 Reverse Transcriptase (HIVRT) is a DNA/RNA-dependent DNA polymerase that plays a central role during viral replication. Our previous studies demonstrated that a nucleotide-induced conformation change governs the HIVRT specificity. Our previous measurements of the rates of polymerization and pyrophosphate release in single turnover experiments with DNA templates showed that pyrophosphate (PPi) dissociation was fast following nucleotide incorporation so that it did not contribute to enzyme specificity (kcat/Km). In the current study, kinetic parameters governing nucleotide incorporation and PPi release were determined using an RNA template. Compared with a DNA template of the same sequence, the rate of chemistry increased by 8-fold (147 vs 18 s−1) while the rate of PPi release decreased to approximate 28 s−1, so that PPi release became the rate-limiting step. During processive nucleotide incorporation, the first nucleotide (TTP) was incorporated at a fast rate (98 s−1), while the incorporation of remaining nucleotides (CGTCG) was much slower with an average rate of 27 s−1, suggesting that sequential incorporation events were limited by the relatively slow PPi release step after incorporation of the prior nucleotide. We then examined the role for PPi release in enzyme specificity. Although PPi release is the rate-determining step it may not be the specificity-determining step, based on our current estimates of the rate of the reversal of the chemistry step. This conclusion could change during misincorporation since PPi release may be extremely slow (∼0.01 s−1) based on our previous data obtained with DNA templates. These studies establish the mechanistic basis for DNA polymerase fidelity during reverse transcription by HIVRT and correct previous estimates that may have underestimated the discrimination factors against mismatched nucleotides because they overlooked the possibility of slow PPi release.