title: C-NMR study on the interaction of medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA.
abstract: The change-transfer interaction in the complex of pig kidney medium-chain acyl-CoA dehydrogenase (MCAD) with acetoacetyl-CoA was investigated by 13C-NMR spectroscopy and molecular orbital treatment. The acyl carbons of acetoacetyl-CoA were separately 13C-labeled and 13C-NMR spectra of the complexes of MCAD with the 13C-labeled acetoacetyl-CoA were measured. Each 13C-carbon atom was observed as a distinct peak and easily distinguished from the protein background. The chemical shift values for free acetoacetyl-CoA were 198.5, 59.9, 208.8, and 32.8 ppm for C(1), C(2), C(3), and C(4), respectively, which shifted to 181.3, 103.4, 192.3, and 29.9 ppm, respectively, when acetoacetyl-CoA was complexed with MCAD. While C(4) underwent a small upfield shift, the other carbons complexed with MCAD. While C(4) underwent a small upfield shift, the other carbons experienced significant shifts; both the C(1) and C(3) carbonyl carbons shifted upfield by about 17 ppm, and the C(2) carbon was observed as a very broad peak at a position shifted downfield by more than 40 ppm. These results were compared with 13C-NMR spectra of the keto-, enol-, and enolate forms of ethyl acetoacetate labeled with 13C at the acyl carbons, and interpreted with reference to the charge-transfer model based on the optimum overlap between the lowest unoccupied molecular orbital (LUMO) of flavin and the highest occupied molecular orbital (HOMO) of the enolate state of the acetoacetyl moiety of acetoacetyl-CoA. The C(2) carbon of acetoacetyl-CoA takes on the sp2 configuration in the bound form, indicating that one of the protons at C(2) of acetoacetyl-CoA is abstracted when bound to MCAD. C(1) = O is substantially polarized in the bound form of acetoacetyl-CoA, implying the presence of a machinery that polarizes this carbonyl group at the binding site, which thereby lowers the pKa value of the alpha-proton at C(2). This machinery is of fundamental importance in the initial step of MCAD catalysis.
authors.affiliation: Department of Biochemistry, Kumamoto University School of Medicine.
authors.forename: R
authors.initials: R
authors.lastname: Miura
authors.forename: Y
authors.initials: Y
authors.lastname: Nishina
authors.forename: S
authors.initials: S
authors.lastname: Fuji
authors.forename: K
authors.initials: K
authors.lastname: Shiga
mesh_terms: D000090:Acetoacetates; D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D005415:Flavins / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D013054:Spectrophotometry, Atomic; D013552:Swine
chemical_list: D000090:Acetoacetates; D000214:Acyl Coenzyme A; D005415:Flavins; C001922:lumiflavin; C010667:acetoacetyl CoA; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; C024840:ethyl acetoacetate