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The Journal of Biochemistry
Year: 2009  |  Volume: 146  |  Issue: 3  |  Page No.: 351 - 357

FT-IR Spectroscopic Studies on the Molecular Mechanism for Substrate Specificity/Activation of Medium-Chain Acyl-CoA Dehydrogenase

Y Nishina, K Sato, H Tamaoki, C Setoyama, R Miura and K. Shiga    

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 measurements. Although 8-NH2-FAD-MCAD did not oxidize acyl-CoA the wavelength of the absorption maximum of the flavin was altered by acyl-CoAs binding. Thus, 8-NH2-FAD-MCAD is one of the attractive materials for investigation of enzyme–substrate (ES) interaction in ES complex (the complex of oxidized MCAD with acyl-CoA). FT-IR difference spectra between non-labelled and [1-13C]-labelled 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 ~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.

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