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. 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.
authors.affiliation: Department of Physics, College of Science, Nanjing Agricultural University, Nanjing 210095, People's Republic of China;
authors.forename: Qun
authors.initials: Q
authors.lastname: Wan
authors.affiliation: University of Tennessee/Oak Ridge National Laboratory Center for Molecular Biophysics, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831;
authors.forename: Jerry M
authors.initials: JM
authors.lastname: Parks
authors.affiliation: Chemistry Department, University of Toledo, Toledo, OH 43606;
authors.forename: B Leif
authors.initials: BL
authors.lastname: Hanson
authors.affiliation: Scientific Activities Division, European Spallation Source, Lund 22100, Sweden;
authors.forename: Suzanne Zoe
authors.initials: SZ
authors.lastname: Fisher
authors.affiliation: Heinz Maier-Leibnitz Zentrum, Technische Universität München, 85748 Garching, Germany;
authors.forename: Andreas
authors.initials: A
authors.lastname: Ostermann
authors.affiliation: Jülich Centre for Neutron Science at Heinz Maier-Leibnitz Zentrum, Forschungszentrum Jülich GmbH, 85747 Garching, Germany;
authors.forename: Tobias E
authors.initials: TE
authors.lastname: Schrader
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831;
authors.forename: David E
authors.initials: DE
authors.lastname: Graham
authors.affiliation: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831.
authors.forename: Leighton
authors.initials: L
authors.lastname: Coates
authors.affiliation: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831.
authors.forename: Paul
authors.initials: P
authors.lastname: Langan
authors.affiliation: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 kovalevskyay@ornl.gov.
authors.forename: Andrey
authors.initials: A
authors.lastname: Kovalevsky
mesh_terms: D055162:Biocatalysis; D002236:Carbohydrate Conformation; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008956:Models, Chemical; D008958:Models, Molecular; D009502:Neutrons; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D055672:Static Electricity; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology
chemical_list: D005656:Fungal Proteins; D006027:Glycosides; D011522:Protons; D018698:Glutamic Acid; D006026:Glycoside Hydrolases
keywords: glycoside hydrolase; macromolecular neutron crystallography; molecular simulations; protonation state; xylanase