W0258

Crystallographic Studies of Medium-Chain Acyl-CoA Dehydrogenase with Mechanism-Based Inhibitors. Ming Wang, Paul A. Hubbard, Rosemary Paschke, and Jung-Ja P. Kim, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226

Medium-chain acyl-CoA dehydrogenase (MCAD) is one of the three soluble flavoproteins that catalyze the first step in mitochondrial ß-oxidation of fatty acids. The enzyme is a homotetramer, with a subunit molecular weight of 45kDa, and contains one FAD per monomer. The substrate/inhibitor binds to the enzyme at the re-face of the FAD in the crevice between the two a-helix domains and the ß-sheet domain of the enzyme. The fatty acyl chain of the thioester substrate is buried inside of the polypeptide and the 3'-AMP moiety of the substrate is close to the surface of the enzyme molecule. The catalytic residue Glu376 abstracts the a proton in the a-ß dehydrogenation reaction catalyzed by the enzyme.

Crystals of several MCAD-inhibitor complexes were prepared by soaking MCAD crystals with various inhibitors, including 2-octynoyl-CoA, methylene-cyclopropylacetyl-CoA (MCPA-CoA) and 5,6-dichloro-4-thia-5- hexanoyl-CoA (DCTH-CoA). The diffraction data sets of complex crystals were collected at 4 ^oC with a Rigaku image plate system to 2.6~2.2 Å resolution depending on the type of inhibitors. The structures of the complexes were solved by the difference Fourier method using phases calculated from the refined native structure. The inhibitors were modeled in and water molecules were added to the structures by using the program TURBO-FRODO. The structures will be refined by using the program

X-PLOR with alternating manual adjustment. Detailed structural comparisons among the structures of these complexes will yield insights into the catalytic mechanism and the exact nature of the chemical reactions of MCAD with these inhibitors.

We thank Dr. Colin Thorpe of University of Delaware for providing us with DCTH-CoA and Dr. Ben Liu of University of Minnesota for MCPA-CoA. The work was supported by an NIH grant GM29076 (J.-J. P. K.).