title: Rate-limiting Pyrophosphate Release by HIV Reverse Transcriptase Improves Fidelity.
abstract: 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 PPirelease 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 PPirelease decreased to approximately 58 s-1so that PPirelease 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 PPirelease step. The accompanying paper shows that slow PPirelease allows polymerization and RNase H to occur at comparable rates. Although PPirelease 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, PPirelease became extremely slow, which we estimated to be ∼0.002 s-1These 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 PPirelease step. Our current estimate of 2.4 × 106is >20-fold greater than estimated previously.
authors: From the University of Texas at Austin, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, Austin, Texas 78712.
authors: An
authors: A
authors: Li
authors: From the University of Texas at Austin, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, Austin, Texas 78712.
authors: Shanzhong
authors: S
authors: Gong
authors: From the University of Texas at Austin, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, Austin, Texas 78712 kajohnson@mail.utexas.edu.
authors: Kenneth A
authors: KA
authors: Johnson
mesh_terms: D011756:Diphosphates / Q000737:chemistry*; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D015497:HIV-1 / Q000201:enzymology*; D016914:Ribonuclease H / Q000737:chemistry*
chemical_list: D011756:Diphosphates; C107241:diphosphoric acid; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H
keywords: DNA polymerase; DNA replication; HIV-1 reverse transcriptase; enzyme kinetics; global data fitting; human immunodeficiency virus (HIV); presteady-state kinetics; pyrophosphate release; transient kinetics