Identification of the Catalytic Base in Long Chain Acyl-CoA Dehydrogenase

• Published in: Biochemistry (Easton) 1994-04, Vol.33 (14), p.4258-4264
• Main Authors: Djordjevic, Snezana, Dong, Yu, Paschke, Rosemary, Frerman, Frank E, Strauss, Arnold W, Kim, Jung-Ja P
• Format: Article
• Language: English
• Subjects: Acyl-CoA Dehydrogenase, Long-Chain - chemistry; Acyl-CoA Dehydrogenase, Long-Chain - genetics; Acyl-CoA Dehydrogenase, Long-Chain - isolation & purification; Acyl-CoA Dehydrogenase, Long-Chain - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding Sites; Biological and medical sciences; Catalysis; Cloning, Molecular; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Humans; Models, Molecular; Molecular Sequence Data; Mutation; Oxidoreductases; Rats; Sequence Homology, Amino Acid; Spectrophotometry, Ultraviolet
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00180a021

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.