CRYSTAL-STRUCTURES OF THE WILD-TYPE AND THE GLU376GLY THR255GLU MUTANT OF HUMAN MEDIUM-CHAIN ACYL-COA DEHYDROGENASE - INFLUENCE OF THE LOCATION OF THE CATALYTIC BASE ON SUBSTRATE-SPECIFICITY/

Crystal structures of the wild type human medium-chain acyl-CoA dehydr ogenase (MCADH) and a double mutant in which its active center base-ar rangement has been altered to that of long chain acyl-CoA dehydrogenas e (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystall ography at 2.75 and 2.4 Angstrom resolution, respectively. The catalyt ic base responsible for the alpha-proton abstraction from the thioeste r substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH n umbering), located over 100 residues away in its primary amino acid se quence. The structures of the mutant complexed with C8-, C12, and C14- CoA have also been determined. The human enzyme structure is essential ly the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few si de chains in the active site cavity. The substrate with chain length l onger than C12 binds to the enzyme in multiple conformations at its om ega-end. Glu255 has two conformations, ''active'' and ''resting'' form s, with the latter apparently stabilized by forming a hydrogen bond wi th Glu99. Both the direction in which Glu255 approaches the C-alpha at om of the substrate and the distance between the Glu255 carboxylate an d the C, atom are different from those of Glu376; these factors are re sponsible for the intrinsic differences in the kinetic properties as w ell as the substrate specificity. Solvent accessible space at the ''mi dsection'' of the active site cavity, where the C-alpha-C-beta bond of the thioester substrate and the isoalloxazine ring of the FAD are loc ated, is larger in the mutant than in the wild type enzyme, implying g reater O-2 accessibility in the mutant which might account for the hig her oxygen reactivity.