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@ARTICLE{Wan:276227,
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},
title = {{D}irect determination of protonation states and
visualization of hydrogen bonding in a glycoside hydrolase
with neutron crystallography},
journal = {Proceedings of the National Academy of Sciences of the
United States of America},
volume = {112},
number = {40},
issn = {1091-6490},
address = {Washington, DC},
publisher = {National Acad. of Sciences},
reportid = {FZJ-2015-06692},
pages = {12384 - 12389},
year = {2015},
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.},
cin = {JCNS (München) ; Jülich Centre for Neutron Science JCNS
(München) ; JCNS-FRM-II / Neutronenstreuung ; JCNS-1},
ddc = {000},
cid = {I:(DE-Juel1)JCNS-FRM-II-20110218 /
I:(DE-Juel1)JCNS-1-20110106},
pnm = {6G15 - FRM II / MLZ (POF3-6G15) / 6G4 - Jülich Centre for
Neutron Research (JCNS) (POF3-623)},
pid = {G:(DE-HGF)POF3-6G15 / G:(DE-HGF)POF3-6G4},
experiment = {EXP:(DE-MLZ)BIODIFF-20140101},
typ = {PUB:(DE-HGF)16},
UT = {WOS:000363125400050},
pubmed = {pmid:26392527},
doi = {10.1073/pnas.1504986112},
url = {https://juser.fz-juelich.de/record/276227},
}