Previous measurements of the rates of polymerization and pyrophosphate release with DNA templates showed that pyrophosphate (PPi) dissociation was fast after nucleotide incorporation so that it did not contribute to enzyme specificity (kcat/Km). Here, 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 up to 10-fold (250 versus 24 s(-1)), whereas the rate of PPi release decreased to approximately 58 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 (152 s(-1)), whereas the rates of incorporation of remaining nucleotides (CGTCG) were much slower with an average rate of 24 s(-1), suggesting that sequential incorporation events were limited by the relatively slow PPi release step. The accompanying paper shows that slow PPi release allows polymerization and RNase H to occur at comparable rates. Although PPi release is the rate-determining step, it is not the specificity-determining step for correct incorporation based on our current estimates of the rate of reversal of the chemistry step (3 s(-1)). In contrast, during misincorporation, PPi release became extremely slow, which we estimated to be ∼0.002 s(-1) These studies establish the mechanistic basis for DNA polymerase fidelity during reverse transcription and provide a free energy profile. We correct previous underestimates of discrimination by including the slow PPi release step. Our current estimate of 2.4 × 10(6) is >20-fold greater than estimated previously.
Rate-limiting Pyrophosphate Release by HIV Reverse Transcriptase Improves Fidelity.