Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

Christian Castro, Eric Smidansky, Kenneth R. Maksimchuk, Jamie J. Arnold, Victoria S. Korneeva, Matthias Götte, William Konigsberg, Craig E. Cameron

Research output: Contribution to journalArticle

107 Citations (Scopus)

Abstract

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 (3Dpol), chemistry is partially (Mg 2+) or completely (Mn2+) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pKa 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.

Original languageEnglish (US)
Pages (from-to)4267-4272
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number11
DOIs
StatePublished - Mar 13 2007

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RNA Replicase
DNA-Directed DNA Polymerase
DNA-Directed RNA Polymerases
Transfer RNA
Protons
Nucleic Acids
Nucleotides
HIV Reverse Transcriptase
Poliovirus
Deuterium
Divalent Cations
Isotopes
Hydroxyl Radical
Catalytic Domain
Equipment and Supplies
Acids
Water
diphosphoric acid

All Science Journal Classification (ASJC) codes

  • General

Cite this

Castro, Christian ; Smidansky, Eric ; Maksimchuk, Kenneth R. ; Arnold, Jamie J. ; Korneeva, Victoria S. ; Götte, Matthias ; Konigsberg, William ; Cameron, Craig E. / Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases. In: Proceedings of the National Academy of Sciences of the United States of America. 2007 ; Vol. 104, No. 11. pp. 4267-4272.
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Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases. / Castro, Christian; Smidansky, Eric; Maksimchuk, Kenneth R.; Arnold, Jamie J.; Korneeva, Victoria S.; Götte, Matthias; Konigsberg, William; Cameron, Craig E.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, No. 11, 13.03.2007, p. 4267-4272.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

AU - Castro, Christian

AU - Smidansky, Eric

AU - Maksimchuk, Kenneth R.

AU - Arnold, Jamie J.

AU - Korneeva, Victoria S.

AU - Götte, Matthias

AU - Konigsberg, William

AU - Cameron, Craig E.

PY - 2007/3/13

Y1 - 2007/3/13

N2 - 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 (3Dpol), chemistry is partially (Mg 2+) or completely (Mn2+) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pKa 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.

AB - 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 (3Dpol), chemistry is partially (Mg 2+) or completely (Mn2+) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pKa 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.

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