W0468

Laue Diffraction of Mitichondrial Aldehyde Dehydrogenase: A Feasibility Study. Samantha J. Perez-Miller and Thomas D. Hurley, Dept. of Biophysics, Indiana Univ. School of Medicine, Indianapolis, IN 46202.

Human mitochondrial aldehyde dehydrogenase (ALDH2) is a key enzyme in ethanol metabolism. After decades of investigation, key questions about the catalytic mechanism remain. One of the most interesting questions arose from initial X-ray structures of aldehyde dehydrogenases complexed with the NAD(P)⁺ cofactor. These structures exhibited evidence for conformational flexibility of the nicotinamide mononucleotide half. We have obtained structures of wild type ALDH2 as well as two inactive mutants in complex with NAD⁺ and NADH which show that the oxidized and reduced cofactor bind in two different conformations. The dominant conformation observed for NAD⁺ is ideal for hydride transfer but would impair deacylation and the only conformation observed for NADH is ideal for deacylation. These structures support the hypothesis that isomerization of the NADH occurs after hydride transfer. In addition, we see no conformational change of the protein backbone when comparing the apo, NAD⁺ and NADH structures. Our next goal is to observe this conformational change using time resolved Laue diffraction. We have collected static Laue data on apo crystals to determine whether this is even feasible. With 220 kDa in the asymmetric unit, typical monochromatic data has > 10,000 spots per image at 2.0 Å. Thus, it is no surprise that significant spatial overlaps limit the resolution and completeness of this data set. Nevertheless, it is clear that with some optimization, time resolved studies with this enzyme will be possible.