Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.
(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:
https://lup.lub.lu.se/record/8035410
- author
- 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
- organization
- publishing date
- 2015
- 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 Academy of Sciences
- external identifiers
-
- pmid:26392527
- wos:000363125400050
- scopus:84943328494
- pmid:26392527
- 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
- 2016-04-01 10:27:36
- date last changed
- 2022-02-02 17:56:54
@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 Academy of 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}},
doi = {{10.1073/pnas.1504986112}},
volume = {{112}},
year = {{2015}},
}