E0007

Spallation Neutron Protein Crystallography: Enzyme Mechanism Studies with D-Xylose Isomerase. B. Leif Hanson*, Paul Langan^, Xinmin Li^, Amy K. Katz^#, Benno P. Schoenborn^, Jenny P. Glusker^#, and Gerard J. Bunick*^$, *UT/ORNL-GST, P.O. Box 2009 MS 8080, Oak Ridge, TN 37831, ^Bioscience Division, LANL, MS M888 Los Alamos, NM 87545, #Fox Chase Cancer Center, Philadelphia, PA 19111, $Life Sciences Division, ORNL, Oak Ridge, TN 37831.

Neutron diffraction data have been measured at the recently commissioned LANSCE macromolecular neutron beamline at Los Alamos for the enzyme D-xylose isomerase (XI). This new neutron beam line provides us with an opportunity to collect diffraction data from higher molecular weight proteins using time-of-flight techniques. arge crystals (1-2 mm) of XI were grown in the EDCAM hardware developed by NASA for microgravity studies. The use of these devices facilitated the month-long H₂O/D₂O exchange process, during which the crystals showed no alterations. One crystal was used with 23 crystal orientations. The data were integrated using the Langan TOF version of D*Trek and normalized and scaled with LAUENORM and SCALA (82,261 reflections, 32,976 unique extending to 1.48 Å resolution with 42% completeness, 70% to 2.0 Å). Results from molecular refinement, currently underway, will be contrasted with structural information from an ultra-high resolution X-ray data structure measured by us. This structure determination of XI reinforces the idea that neutron diffraction is unsurpassed as a method for accurately locating the positions of hydrogen atoms. This represents the initial stage of a long-term study using inhibitor complexes and differing metal ions to help determine the mechanism of XI by locating key hydrogen atoms.

Supported by Grants CA-10925, CA-06927 and GM-29818 from the National Institutes of Health, U. S. Public Health Service; NAG8-1826 from NASA; Laboratory Directed Research and Development Program ORNL, managed by UT-Battelle, LLC for the U. S. DOE under Contract No. DE-AC05-00OR22725; DOE Office of Science, Office of Basic Energy Sciences, and Office of Biological and Environmental Research (LANL and access to the Protein Crystallography Station at LANSCE).