Rate-limiting Pyrophosphate Release by HIV Reverse Transcriptase Improves Fidelity.

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 × 106 is >20-fold greater than estimated previously.