ArticleProceedings of the National Academy of Sciences of the United States of America2015
Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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Who cites it
25 citing papers in PubMed, 43 citations in OpenAlex.
- Exploration of Alkaliphilic Bacteria Alkalitalea saponilacus Identifies Glycoside Hydrolases With Biotechnological Potential.Microbial biotechnology · 2026Article
- Neutron crystallography of the covalent intermediate of β-glucosidase reveals remodeling of the catalytic center.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- "Seeing Is Believing": How Neutron Crystallography Informs Enzyme Mechanisms by Visualizing Unique Water Species.Biology · 2024Review
- Development of Tunable Mechanism-Based Carbasugar Ligands that Stabilize Glycoside Hydrolases through the Formation of Transient Covalent Intermediates.ACS catalysis · 2024Article
- Neutron diffraction from a microgravity-grown crystal reveals the active site hydrogens of the internal aldimine form of tryptophan synthase.Cell reports. Physical science · 2024Article
- Neutron crystallographic refinement with REFMAC5 from the CCP4 suite.Acta crystallographica. Section D, Structural biology · 2023Article
- Investigating the effect of substrate binding on the catalytic activity of xylanase.Applied microbiology and biotechnology · 2023Article
- Description of peptide bond planarity from high-resolution neutron crystallography.Biophysics and physicobiology · 2023Article
- Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase.Chemical science · 2022Article
- Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases.Biomolecules · 2022Article
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- Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid-Base Catalysis.ACS catalysis · 2021Article
- Configurational Entropy of Folded Proteins and Its Importance for Intrinsically Disordered Proteins.International journal of molecular sciences · 2021Article
- Studying the Role of a Single Mutation of a Family 11 Glycoside Hydrolase Using High-Resolution X-ray Crystallography.The protein journal · 2020Article
- Maize WI5 encodes an endo-1,4-β-xylanase required for secondary cell wall synthesis and water transport in xylem.Journal of integrative plant biology · 2020Article
- Proton transfer and drug binding details revealed in neutron diffraction studies of wild-type and drug resistant HIV-1 protease.Methods in enzymology · 2020Article
- Titration of ionizable groups in proteins using multiple neutron data sets from a single crystal: application to the small GTPase Ras.Acta crystallographica. Section F, Structural biology communications · 2019Article
- Neutron and X-ray crystal structures of Lactobacillus brevis alcohol dehydrogenase reveal new insights into hydrogen-bonding pathways.Acta crystallographica. Section F, Structural biology communications · 2018Article
- Improving the accuracy and resolution of neutron crystallographic data by three-dimensional profile fitting of Bragg peaks in reciprocal space.Acta crystallographica. Section D, Structural biology · 2018Article
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Authors and funding
10 authors at 7 institutions in 4 countries.
Funding
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.
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