Journal of Biochemistry Advance Access published online on May 26, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp077
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
FT-IR Spectroscopic Studies on the Molecular Mechanism for Substrate Specificity/Activation of Medium-Chain Acyl-CoA Dehydrogenase
1Department of Physiology, School of Health Sciences, Kumamoto University, Kuhonji, Kumamoto 862-0976, Departments of 2Molecular Physiology and 3Molecular Enzymology, Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto 860-8556, and 4Department of Nursing, Kyushu University of Nursing and Social Welfare, Tomio, Tamana, Kumamoto 865-0062
To whom correspondence should be addressed: Dr. Nishina, Yasuzo,E-mail: nishina{at}kumamoto-u.ac.jp
Received April 9, 2009; Accepted May 11, 2009
 |
Abstract |
|---|
The interactions of acyl-CoA with medium-chain acyl-CoA dehydrogenases (MCADs) reconstituted with artificial FADs, i.e., 8-CN-, 7,8-Cl2-, 8-Cl-, 8-OCH3-, and 8-NH2-FAD were investigated by UV-visible absorption and FT-IR measurments. 8-NH2-FAD-MCAD bound acyl-CoA but did not oxidize acyl-CoA and the wavelength of the absorption maximum of the flavin was altered by acyl-CoAs. Thus 8-NH2-FAD-MCAD is one of attractive materials for investigation of enzyme-substrate interaction in ES-complex (the complex of oxidized MCAD with acyl-CoA). FT-IR difference spectra between non-labeled and [1-13C]-labeled acyl-CoA free in solution and bound to oxidized 8-NH2-FAD-MCAD were obtained. The broad 1668-cm-1 band of free octanoyl-CoA assigned to the C(1)=O stretching vibration appeared as a sharp signal at 1626 cm-1 in the case of the complex. The downward shift indicates a large polarization of C(1)=O, and the sharpness suggests that the orientation of the C(1)=O in the active site cavity is fairly limited. The hydrogen-bond enthalpy change responsible for the polarization on the transfer of the substrate from aqueous solution to the active site of MCAD was estimated to be ca. 15 kcal/mol. The 1626-cm-1 band is noticeably weakened in the case of acyl-CoA with acyl-chains longer than C12 which are poor substrates for MCAD, suggesting that C(1)=O is likely to exist in multiple orientations in the active site cavity, whence the band becomes obscured. A band identical to that of bound C8-CoA was observed in the case of C4-CoA which is a poor substrate, indicating the strong hydrogen bond at C(1)=O.
Key Words: artificial flavin, enzyme-substrate complex, FTIR spectroscopy, hydrogen bond, medium-chain acyl-CoA dehydrogenase.