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

Wan, Qun; Parks, Jerry M; Hanson, B Leif; Fisher, Zoe LU ; Ostermann, Andreas; Schrader, Tobias E; Graham, David E; Coates, Leighton; Langan, Paul and Kovalevsky, Andrey (2015) In Proceedings of the National Academy of Sciences 112(40). p.12384-12389
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. However, 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... (More)
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. However, 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. 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. 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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Proceedings of the National Academy of Sciences
volume
112
issue
40
pages
12384 - 12389
publisher
National Acad Sciences
external identifiers
  • pmid:26392527
  • wos:000363125400050
  • scopus:84943328494
ISSN
1091-6490
DOI
10.1073/pnas.1504986112
language
English
LU publication?
yes
id
a2db4f1e-e27f-403d-b894-2fa831ad9f54 (old id 8035410)
date added to LUP
2015-10-28 07:50:19
date last changed
2017-10-29 03:11:26
@article{a2db4f1e-e27f-403d-b894-2fa831ad9f54,
  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. However, 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. 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. 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.},
  author       = {Wan, Qun and Parks, Jerry M and Hanson, B Leif and Fisher, Zoe and Ostermann, Andreas and Schrader, Tobias E and Graham, David E and Coates, Leighton and Langan, Paul and Kovalevsky, Andrey},
  issn         = {1091-6490},
  language     = {eng},
  number       = {40},
  pages        = {12384--12389},
  publisher    = {National Acad Sciences},
  series       = {Proceedings of the National Academy of Sciences},
  title        = {Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.},
  url          = {http://dx.doi.org/10.1073/pnas.1504986112},
  volume       = {112},
  year         = {2015},
}