NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase

C. Viragh, Thomas K Harris, P. M. Reddy, M. A. Massiah, A. S. Mildvan, I. M. Kovach

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by 1H NMR at 600 MHz and 25-37 °C. The downfield region of the 1H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, δ = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor φ = 0.72 ± 0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of δ with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64 ± 0.04 Å, in agreement with the length of 2.62 ± 0.02 Å independently obtained from φ. The addition of a 3-fold excess of m-(N,N,N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift δ = 18.1 ppm and a D/H fractionation factor φ = 0.63 ± 0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62 ± 0.04 Å from δ and 2.59 ± 0.03 Å from φ. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60 ± 0.22 and 2.66 ± 0.28 Å. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.

Original languageEnglish
Pages (from-to)16200-16205
Number of pages6
JournalBiochemistry
Volume39
Issue number51
DOIs
StatePublished - Dec 26 2000
Externally publishedYes

Fingerprint

Cholinesterases
Hydrogen
Catalytic Domain
Hydrogen bonds
Nuclear magnetic resonance
Butyrylcholinesterase
Histidine
Chemical shift
Fractionation
Dealkylation
Paraoxon
Serine Proteases
Hydrogen Bonding
Acetylcholinesterase
Biochemistry
Catalysis
Serine
Bond length
Horses
Protons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Viragh, C., Harris, T. K., Reddy, P. M., Massiah, M. A., Mildvan, A. S., & Kovach, I. M. (2000). NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase. Biochemistry, 39(51), 16200-16205. https://doi.org/10.1021/bi0022644

NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase. / Viragh, C.; Harris, Thomas K; Reddy, P. M.; Massiah, M. A.; Mildvan, A. S.; Kovach, I. M.

In: Biochemistry, Vol. 39, No. 51, 26.12.2000, p. 16200-16205.

Research output: Contribution to journalArticle

Viragh, C, Harris, TK, Reddy, PM, Massiah, MA, Mildvan, AS & Kovach, IM 2000, 'NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase', Biochemistry, vol. 39, no. 51, pp. 16200-16205. https://doi.org/10.1021/bi0022644
Viragh, C. ; Harris, Thomas K ; Reddy, P. M. ; Massiah, M. A. ; Mildvan, A. S. ; Kovach, I. M. / NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase. In: Biochemistry. 2000 ; Vol. 39, No. 51. pp. 16200-16205.
@article{4464080fb69047ff8ee127093d5a04d1,
title = "NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase",
abstract = "Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by 1H NMR at 600 MHz and 25-37 °C. The downfield region of the 1H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, δ = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor φ = 0.72 ± 0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of δ with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64 ± 0.04 {\AA}, in agreement with the length of 2.62 ± 0.02 {\AA} independently obtained from φ. The addition of a 3-fold excess of m-(N,N,N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift δ = 18.1 ppm and a D/H fractionation factor φ = 0.63 ± 0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62 ± 0.04 {\AA} from δ and 2.59 ± 0.03 {\AA} from φ. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60 ± 0.22 and 2.66 ± 0.28 {\AA}. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.",
author = "C. Viragh and Harris, {Thomas K} and Reddy, {P. M.} and Massiah, {M. A.} and Mildvan, {A. S.} and Kovach, {I. M.}",
year = "2000",
month = "12",
day = "26",
doi = "10.1021/bi0022644",
language = "English",
volume = "39",
pages = "16200--16205",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "51",

}

TY - JOUR

T1 - NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase

AU - Viragh, C.

AU - Harris, Thomas K

AU - Reddy, P. M.

AU - Massiah, M. A.

AU - Mildvan, A. S.

AU - Kovach, I. M.

PY - 2000/12/26

Y1 - 2000/12/26

N2 - Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by 1H NMR at 600 MHz and 25-37 °C. The downfield region of the 1H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, δ = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor φ = 0.72 ± 0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of δ with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64 ± 0.04 Å, in agreement with the length of 2.62 ± 0.02 Å independently obtained from φ. The addition of a 3-fold excess of m-(N,N,N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift δ = 18.1 ppm and a D/H fractionation factor φ = 0.63 ± 0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62 ± 0.04 Å from δ and 2.59 ± 0.03 Å from φ. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60 ± 0.22 and 2.66 ± 0.28 Å. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.

AB - Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by 1H NMR at 600 MHz and 25-37 °C. The downfield region of the 1H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, δ = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor φ = 0.72 ± 0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of δ with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64 ± 0.04 Å, in agreement with the length of 2.62 ± 0.02 Å independently obtained from φ. The addition of a 3-fold excess of m-(N,N,N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift δ = 18.1 ppm and a D/H fractionation factor φ = 0.63 ± 0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62 ± 0.04 Å from δ and 2.59 ± 0.03 Å from φ. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60 ± 0.22 and 2.66 ± 0.28 Å. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.

UR - http://www.scopus.com/inward/record.url?scp=0034719145&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034719145&partnerID=8YFLogxK

U2 - 10.1021/bi0022644

DO - 10.1021/bi0022644

M3 - Article

VL - 39

SP - 16200

EP - 16205

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 51

ER -