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Title: Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Abstract

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. Yet, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. Here, we performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. Lastly, these findings shed light on the initial stagemore » of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.« less

Authors:
 [1]; ORCiD logo [2];  [3];  [4];  [5];  [6]; ORCiD logo [7];  [7]; ORCiD logo [7]; ORCiD logo [7]
  1. Nanjing Agricultural Univ., Nanjing (People's Republic of China)
  2. Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  3. Univ. of Toledo, Toledo, OH (United States)
  4. European Spallation Source, Lund (Sweden)
  5. Technische Univ. Munchen, Garching (Germany)
  6. Forschungszentrum Julich GmbH, Garching (Germany)
  7. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Laboratory Directed Research and Development (LDRD) Program
OSTI Identifier:
1265915
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 40; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; glycoside hydrolase; protonation state; macromolecular neutron; crystallography; xylanase; molecular simulations

Citation Formats

Wan, Qun, Parks, Jerry M., Hanson, B. Leif, Fisher, Suzanne Zoe, Ostermann, Andreas, Schrader, Tobias E., Graham, David E., Coates, Leighton, Langan, Paul, and Kovalevsky, Andrey. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography. United States: N. p., 2015. Web. doi:10.1073/pnas.1504986112.
Wan, Qun, Parks, Jerry M., Hanson, B. Leif, Fisher, Suzanne Zoe, Ostermann, Andreas, Schrader, Tobias E., Graham, David E., Coates, Leighton, Langan, Paul, & Kovalevsky, Andrey. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography. United States. doi:10.1073/pnas.1504986112.
Wan, Qun, Parks, Jerry M., Hanson, B. Leif, Fisher, Suzanne Zoe, Ostermann, Andreas, Schrader, Tobias E., Graham, David E., Coates, Leighton, Langan, Paul, and Kovalevsky, Andrey. Mon . "Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography". United States. doi:10.1073/pnas.1504986112. https://www.osti.gov/servlets/purl/1265915.
@article{osti_1265915,
title = {Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography},
author = {Wan, Qun and Parks, Jerry M. and Hanson, B. Leif and Fisher, Suzanne Zoe and Ostermann, Andreas and Schrader, Tobias E. and Graham, David E. and Coates, Leighton and Langan, Paul and Kovalevsky, Andrey},
abstractNote = {Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. Yet, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. Here, we performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. Lastly, these findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.},
doi = {10.1073/pnas.1504986112},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 40,
volume = 112,
place = {United States},
year = {2015},
month = {9}
}

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    Improving the accuracy and resolution of neutron crystallographic data by three-dimensional profile fitting of Bragg peaks in reciprocal space
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    • Sullivan, Brendan; Archibald, Rick; Langan, Patricia S.
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