title: FT-IR spectroscopic studies on the molecular mechanism for substrate specificity/activation of medium-chain acyl-CoA dehydrogenase.
abstract: 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.
authors.affiliation: Department of Physiology, School of Health Sciences, Kumamoto University, Kuhonji, Kumamoto 862-0976, Japan. nishina@kumamoto-u.ac.jp
authors.forename: Yasuzo
authors.initials: Y
authors.lastname: Nishina
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Sato
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Tamaoki
authors.forename: Chiaki
authors.initials: C
authors.lastname: Setoyama
authors.forename: Retsu
authors.initials: R
authors.lastname: Miura
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Shiga
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D042964:Acyl-CoA Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D002247:Carbon Isotopes; D020134:Catalytic Domain; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D011485:Protein Binding; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D013552:Swine
chemical_list: C544757:8-amino-FAD; D000214:Acyl Coenzyme A; D002247:Carbon Isotopes; C034681:octanoyl-coenzyme A; D005182:Flavin-Adenine Dinucleotide; D042964:Acyl-CoA Dehydrogenase