IDENTIFICATION OF THE CATALYTIC BASE IN LONG-CHAIN ACYL-COA DEHYDROGENASE

We have used molecular modeling and site-directed mutagenesis to ident ify the catalytic residues of human long chain acyl-CoA dehydrogenase. Among the acyl-CoA dehydrogenases, a family of flavoenzymes involved in beta-oxidation of fatty acids, only the three-dimensional structure of the medium chain fatty acid specific enzyme from pig liver has bee n determined (Kim, J.-J. P., Wang, M., & Paschke, R. (1993) Proc. Natl . Acad. Sci. U.S.A. 90, 7523-7527). Despite the overall sequence homol ogy, the catalytic residue (E376) of medium chain acyl-CoA dehydrogena se is not conserved in isovaleryl- and long chain acyl-CoA dehydrogena ses. A molecular model of human long chain acyl-CoA dehydrogenase was derived using atomic coordinates determined by X-ray diffraction studi es of the pig medium chain specific enzyme, interactive graphics, and molecular mechanics calculations. The model suggests that E261 functio ns as the catalytic base in the long-chain dehydrogenase. An altered d ehydrogenase in which E261 was replaced by a glutamine was constructed , expressed, purified, and characterized. The mutant enzyme exhibited less than 0.02% of the wild-type activity. These data strongly suggest that E261 is the base that abstracts the alpha-proton of the acyl-CoA substrate in the catalytic pathway of this dehydrogenase.