Locating active-site hydrogen atoms in D-xylose isomerase: time-of-flight neutron diffraction

Proceedings of the National Academy of Sciences of the United States of America
Amy K KatzGerard J Bunick

Abstract

Time-of-flight neutron diffraction has been used to locate hydrogen atoms that define the ionization states of amino acids in crystals of D-xylose isomerase. This enzyme, from Streptomyces rubiginosus, is one of the largest enzymes studied to date at high resolution (1.8 A) by this method. We have determined the position and orientation of a metal ion-bound water molecule that is located in the active site of the enzyme; this water has been thought to be involved in the isomerization step in which D-xylose is converted to D-xylulose or D-glucose to D-fructose. It is shown to be water (rather than a hydroxyl group) under the conditions of measurement (pH 8.0). Our analyses also reveal that one lysine probably has an -NH(2)-terminal group (rather than NH(3)(+)). The ionization state of each histidine residue also was determined. High-resolution x-ray studies (at 0.94 A) indicate disorder in some side chains when a truncated substrate is bound and suggest how some side chains might move during catalysis. This combination of time-of-flight neutron diffraction and x-ray diffraction can contribute greatly to the elucidation of enzyme mechanisms.

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Citations

Mar 7, 2013·Applied Microbiology and Biotechnology·Hajer Ben HlimaNushin Aghajari
Jan 13, 2009·Proceedings of the National Academy of Sciences of the United States of America·Marc-Michael BlumJulian C-H Chen
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Related Concepts

Glucose isomerase
Catalysis
Polymorphism, Crystallization
Glucose, (beta-D)-Isomer
Hydrogen
Substrate Specificity
Tertiary Protein Structure
Aldose-Ketose Isomerases
Neutron Diffraction

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