4XPV

Neutron and X-ray structure analysis of xylanase: N44D at pH6

4XPV の概要

• エントリーDOI: 10.2210/pdb4xpv/pdb
• 分子名称: Endo-1,4-beta-xylanase 2, IODIDE ION (3 entities in total)
• 機能のキーワード: jelly roll, hydrolase
• 由来する生物種: Hypocrea jecorina
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 21109.03

構造登録者

Wan, Q.,Park, J.M.,Riccardi, D.M.,Hanson, L.B.,Fisher, Z.,Smith, J.C.,Ostermann, A.,Schrader, T.,Graham, D.E.,Coates, L.,Langan, P.,Kovalevsky, A.Y. (登録日: 2015-01-18, 公開日: 2015-09-30, 最終更新日: 2023-09-27)

主引用文献

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

実験手法

NEUTRON DIFFRACTION (2 Å)
X-RAY DIFFRACTION (1.7 Å)