Rate-limiting Pyrophosphate Release by HIV Reverse Transcriptase Improves Fidelity

摘要: Previous measurements of the rates polymerization and pyrophosphate release with DNA templates showed that (PPi) dissociation was fast after nucleotide incorporation so it did not contribute to enzyme specificity (kcat/Km). Here, kinetic parameters governing PPi were determined using an RNA template. Compared a template same sequence, rate chemistry increased by up 10-fold (250 versus 24 s-1), whereas decreased approximately 58 s-1 became rate-limiting step. During processive incorporation, first (TTP) incorporated at (152 remaining nucleotides (CGTCG) much slower average s-1, suggesting sequential events limited relatively slow The accompanying paper shows allows RNase H occur comparable rates. Although is rate-determining step, specificity-determining step for correct based on our current estimates reversal (3 s-1). In contrast, during misincorporation, extremely slow, which we estimated be ∼0.002 These studies establish mechanistic basis polymerase fidelity reverse transcription provide free energy profile. We previous underestimates discrimination including Our estimate 2.4 × 106 >20-fold greater than previously.