4S2G

Joint X-ray/neutron structure of Trichoderma reesei xylanase II at pH 5.8

Summary for 4S2G

• Entry DOI: 10.2210/pdb4s2g/pdb
• Related: 4S2D 4S2F 4S2H
• Descriptor: Endo-1,4-beta-xylanase 2, IODIDE ION (3 entities in total)
• Functional Keywords: glycoside hydrolase, hydrolase
• Biological source: Trichoderma reesei
• Total number of polymer chains: 1
• Total formula weight: 20965.34

Authors

Kovalevsky, A.,Wan, Q.,Langan, P. (deposition date: 2015-01-20, release date: 2015-09-23, Last modification date: 2024-11-20)

Primary citation

Wan, Q.,Parks, J.M.,Hanson, B.L.,Fisher, S.Z.,Ostermann, A.,Schrader, T.E.,Graham, D.E.,Coates, L.,Langan, P.,Kovalevsky, A.
Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.
Proc.Natl.Acad.Sci.USA, 112:12384-12389, 2015

PubMed 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.

PubMed: 26392527
DOI: 10.1073/pnas.1504986112

Experimental method

NEUTRON DIFFRACTION (2 Å)
X-RAY DIFFRACTION (1.6 Å)