Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography
| Autor: | Leighton Coates, Andreas Ostermann, S. Z. Fisher, David E. Graham, Tobias E. Schrader, B. Leif Hanson, Jerry M. Parks, Qun Wan, Andrey Kovalevsky, Paul Langan |
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| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Glycoside Hydrolases Stereochemistry Static Electricity Glutamic Acid Protonation Crystallography X-Ray Substrate Specificity Catalysis Fungal Proteins Residue (chemistry) Catalytic Domain Hydrolase Carbohydrate Conformation Glycoside hydrolase Glycosides Neutrons Trichoderma chemistry.chemical_classification Multidisciplinary biology Hydrogen bond Hydrolysis Temperature Active site Hydrogen Bonding Glycosidic bond Biological Sciences Hydrogen-Ion Concentration Protein Structure Tertiary Crystallography Models Chemical chemistry Biocatalysis biology.protein Protons Protein Binding |
| Zdroj: | Proceedings of the National Academy of Sciences. 112:12384-12389 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1504986112 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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