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, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

doi:10.1073/pnas.1504986112

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

Journal Article · 20 September 2015 · Proceedings of the National Academy of Sciences of the United States of America

DOI: https://doi.org/10.1073/pnas.1504986112 · OSTI ID:1265915

Wan, Qun ^[1];

^[2]; Hanson, B. Leif ^[3]; Fisher, Suzanne Zoe ^[4]; Ostermann, Andreas ^[5]; Schrader, Tobias E. ^[6];

^[7]; Coates, Leighton ^[7];

^[7];

^[7]

Nanjing Agricultural Univ., Nanjing (People's Republic of China)
Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Univ. of Toledo, Toledo, OH (United States)
European Spallation Source, Lund (Sweden)
Technische Univ. Munchen, Garching (Germany)
Forschungszentrum Julich GmbH, Garching (Germany)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

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 pK_a 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 pK_a values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pK_a 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.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: ORNL Program Development; USDOE

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1265915

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 40 Vol. 112; ISSN 0027-8424

Publisher:: National Academy of Sciences, Washington, DC (United States)Copyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
crystallography
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Title: Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

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