Transition state structure of E. coli tRNA-specific adenosine deaminase

Minkui Luo, Vern L. Schramm

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single TRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNAArg2 was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1′-3H], [5′-3H2], [1′-14C], [6- 13C], [6-15N], [6-13C, 6-15N] and [1-15N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-13C]-, [6-15N]-, [1-15N]-, [1′-3H]-, [5′-3H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late S NAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-15N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 Å in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 Å, respectively. The large inverse [5′-3H] KIE of -3.7% and modest normal [1′-3H] KIE of 1.4% indicate that significant ribosyl 5′-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes.

Original languageEnglish (US)
Pages (from-to)2649-2655
Number of pages7
JournalJournal of the American Chemical Society
Volume130
Issue number8
DOIs
StatePublished - Feb 27 2008

Fingerprint

Adenosine Deaminase
Transfer RNA
Isotopes
Escherichia coli
Kinetics
Hydroxyl Radical
Cytidine Deaminase
RNA Editing
Inosine
Deamination
Nucleophiles
Proton transfer
Protonation
Substrates
Plasmodium falciparum
RNA
Catalysis
Codon
Adenosine
Protons

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Transition state structure of E. coli tRNA-specific adenosine deaminase. / Luo, Minkui; Schramm, Vern L.

In: Journal of the American Chemical Society, Vol. 130, No. 8, 27.02.2008, p. 2649-2655.

Research output: Contribution to journalArticle

@article{7f46723e4a9a48d4a7cb6d6b05bb23dc,
title = "Transition state structure of E. coli tRNA-specific adenosine deaminase",
abstract = "Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single TRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNAArg2 was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1′-3H], [5′-3H2], [1′-14C], [6- 13C], [6-15N], [6-13C, 6-15N] and [1-15N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-13C]-, [6-15N]-, [1-15N]-, [1′-3H]-, [5′-3H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late S NAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-15N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 {\AA} in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 {\AA}, respectively. The large inverse [5′-3H] KIE of -3.7{\%} and modest normal [1′-3H] KIE of 1.4{\%} indicate that significant ribosyl 5′-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes.",
author = "Minkui Luo and Schramm, {Vern L.}",
year = "2008",
month = "2",
day = "27",
doi = "10.1021/ja078008x",
language = "English (US)",
volume = "130",
pages = "2649--2655",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "8",

}

TY - JOUR

T1 - Transition state structure of E. coli tRNA-specific adenosine deaminase

AU - Luo, Minkui

AU - Schramm, Vern L.

PY - 2008/2/27

Y1 - 2008/2/27

N2 - Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single TRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNAArg2 was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1′-3H], [5′-3H2], [1′-14C], [6- 13C], [6-15N], [6-13C, 6-15N] and [1-15N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-13C]-, [6-15N]-, [1-15N]-, [1′-3H]-, [5′-3H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late S NAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-15N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 Å in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 Å, respectively. The large inverse [5′-3H] KIE of -3.7% and modest normal [1′-3H] KIE of 1.4% indicate that significant ribosyl 5′-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes.

AB - Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single TRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNAArg2 was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1′-3H], [5′-3H2], [1′-14C], [6- 13C], [6-15N], [6-13C, 6-15N] and [1-15N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-13C]-, [6-15N]-, [1-15N]-, [1′-3H]-, [5′-3H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late S NAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-15N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 Å in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 Å, respectively. The large inverse [5′-3H] KIE of -3.7% and modest normal [1′-3H] KIE of 1.4% indicate that significant ribosyl 5′-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes.

UR - http://www.scopus.com/inward/record.url?scp=39749128713&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=39749128713&partnerID=8YFLogxK

U2 - 10.1021/ja078008x

DO - 10.1021/ja078008x

M3 - Article

VL - 130

SP - 2649

EP - 2655

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 8

ER -