| 19470521 |
Nishina Y, Sato K, Tamaoki H, Setoyama C, Miura R, Shiga K: FT-IR spectroscopic studies on the molecular mechanism for substrate specificity/activation of medium-chain acyl-CoA dehydrogenase. J Biochem. 2009 Sep;146(3):351-7. Epub 2009 May 26.
The interactions of acyl-CoA with medium-chain acyl-CoA dehydrogenases (MCADs) reconstituted with artificial FADs-i.e. 8-CN-, 7,8-Cl (2)-, 8-Cl-, 8-OCH (3)- and 8-NH (2)-FAD-were investigated by UV-visible absorption and FT-IR measurements. Although 8-NH (2)-FAD-MCAD did not oxidize acyl-CoA the wavelength of the absorption maximum of the flavin was altered by acyl-CoAs binding. Thus, 8-NH (2)-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-(13) C]-labelled acyl-CoA free in solution and bound to oxidized 8-NH (2)-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 approximately 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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