Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases
| Autor: | Jamie J. Arnold, Craig E. Cameron, Kenneth R. Maksimchuk, Victoria S. Korneeva, Christian Castro, Matthias Götte, William H. Konigsberg, Eric D. Smidansky |
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| Rok vydání: | 2007 |
| Předmět: |
DNA polymerase
Stereochemistry Protonation DNA-Directed DNA Polymerase Catalysis chemistry.chemical_compound Deprotonation RNA polymerase Escherichia coli Magnesium Polymerase Manganese Binding Sites Multidisciplinary biology DNA DNA-Directed RNA Polymerases Biological Sciences Hydrogen-Ion Concentration RNA-Dependent RNA Polymerase Kinetics Poliovirus Biochemistry chemistry Phosphodiester bond biology.protein Nucleic acid Nucleic Acid Conformation RNA Steady state (chemistry) Protons |
| Zdroj: | Proceedings of the National Academy of Sciences. 104:4267-4272 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.0608952104 |
| Popis: | The rate-limiting step for nucleotide incorporation in the pre-steady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D pol ), chemistry is partially (Mg 2+ ) or completely (Mn 2+ ) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK a values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3′-hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleotidyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3′-OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure. |
| Databáze: | OpenAIRE |
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