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With its international funding and expertise the Institut Laue-Langevin (ILL) offers scientists and industry the world's leading facility in neutron science and technology. From its Grenoble site in the south-east of France the Institute operates the most intense neutron source on earth.

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21 June 2011 10:00 Age: 338 days

Identification of the Elusive Hydronium Ion Replacing a Proton in an Enzyme

A  collaborative effort between scientists at the ILL, Los Alamos National Laboratory, Keele University, ISIS, and the University of Toledo has resulted in the remarkable observation of hydroxonium ions that are thought to be involved in proton transfer during enzyme-catalysed reactions.

The work, which used ILL's D19 diffractometer, exploits the remarkable ability of neutron diffraction to visualise hydrogen atoms and therefore to be able to distinguish between hydroxonium ions and water molecules - this is typically impossible using X-ray crystallography.  The enzyme studied was xylose isomerase - this is an enzyme of high biological and biotechnological significance, and one where changes in the location of hydrogen atoms are crucial to catalytic activity that is associated with converting D-xylose to D-xylulose. It turns out that hydroxonium ions replace metal ions and may be involved in the templating specific sites for the binding of metal ions. The work explains how the activity of the enzyme decreases dramatically at low pH.


This is the first direct visualisation of a hydroxonium ion in a biological system and has important implications for an understanding of the role of these ions in other macromolecular systems.  This is an area where neutron crystallography instruments such as D19 and LADI-III are likely to provide key information in the future, and where the strong synergy between advanced capabilities of neutrons sources such as the ILL and synchrotron radiation sources such as ESRF together offer insights that are beyond either approach alone.


 

Figure 1. The M1 metal-binding site of the d-xylose isomerase active
site: 2FO - FC (violet) and omit FO - FC (green) positive neutron scattering
density maps of the active site of d-xylose isomerase showing
a) D3O+ at pH 7.7 and b) D+ at pH 5.9; H bonds are shown as blue
dotted lines. Coordinates and structure factors have been deposited in
the Protein Data Bank with the accession codes 3KCJ (apo-XI, pH 7.7),
3QZA (apo-XI, pH 5.9), and 3QYS (XI–0.6Ni2+ complex, pH 5.8

Figure 2. Superposition of the M1 metal-binding sites of the structures
of the apo form of d-xylose isomerase at the pH values of 7.7 (yellow
carbon atoms) and 5.9 (blue carbon atoms) showing the collapse of
side chains in this site by more than 1 Angström in terms of the differences in
O···O separation. These structural changes are significant considering
the coordinate errors (diffraction-data precision indicator, DPI) in both
structures of 0.18 Angström.


Angewandte Chemie, 2011, 123, 1 – 4.

Andrey Y. Kovalevsky, B. L. Hanson, S. A. Mason, T. Yoshida, S. Z. Fisher, M. Mustyakimov, V. T. Forsyth, M. P. Blakeley, D. A. Keen, Paul Langan
Angewandte Chemie

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