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Hydrogen Location in Stages of an Enzyme-Catalyzed Reaction: Time-of-Flight Neutron Structure of d-Xylose Isomerase with Bound d-Xylulose^†^‡

Supporting Info

Andrey Y. Kovalevsky^§, Amy K. Katz, H. L. Carrell, Leif Hanson, Marat Mustyakimov^§, S. Zoe Fisher^§, Leighton Coates^§^@, Benno P. Schoenborn^§, Gerard J. Bunick⁺^#, Jenny P. Glusker and Paul Langan*^§

M888, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111-2497, Chemistry Department, University of Toledo, Toledo, Ohio 43606, and Chemistry Department, University of Tennessee, Knoxville, Tennessee 37996

Biochemistry, 2008, 47 (29), pp 7595–7597

DOI: 10.1021/bi8005434

Publication Date (Web): June 26, 2008

†

A.K.K., H.L.C., M.M., P.L., and J.P.G. were supported by grants from the National Institutes of Health (CA06927, CA10925, and GM071939). The PCS is funded by the Office of Biological and Environmental Research of the U.S. Department of Energy.

‡

Coordinates and structure factors have been deposited in the Protein Data Bank as entry 3CWH.

Los Alamos National Laboratory.

Fox Chase Cancer Center.

University of Toledo.

Current address: Spallation Neutron Source, Oak Ridge National Laboratory, Oak Ridge, TN 37831.

University of Tennessee.

Recently deceased.

* To whom correspondence should be addressed. Phone: (505) 665-8125. Fax: (505) 665-3024. E-mail: langan_paul@lanl.gov.

Abstract

The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme d-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, d-xylulose, shows, unexpectedly, that O5 of d-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in the proximity of C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.

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