Identification of the catalytic base in long chain acyl-CoA dehydrogenase.
We have used molecular modeling and site-directed mutagenesis to identify 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 been determined (Kim, J.-J.P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527). Despite the overall sequence homology, the catalytic residue (E376) of medium chain acyl-CoA dehydrogenase is not conserved in isovaleryl- and long chain acyl-CoA dehydrogenases. A molecular model of human long chain acyl-CoA dehydrogenase was derived using atomic coordinates determined by X-ray diffraction studies of the pig medium chain specific enzyme, interactive graphics, and molecular mechanics calculations. The model suggests that E261 functions as the catalytic base in the long-chain dehydrogenase. An altered dehydrogenase 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.
Djordjevic
S
S
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Dong
Y
Y
Paschke
R
R
Frerman
F E
FE
Strauss
A W
AW
Kim
J J
JJ
D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D044942:Acyl-CoA Dehydrogenase, Long-Chain