caa a22 4500 397558406 CHVBK 20180308164809.0 cr unu---uuuuu 161202e199603 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021271 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021271 13C-NMR Study on the Interaction of Medium-Chain Acyl-CoA Dehydrogenase with Acetoacetyl-CoA [Elektronische Daten] [Retsu Miura, Yasuzo Nishina, Shigeru Fujii, Kiyoshi Shiga] The charge-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 the13C-labeled acetoace-tyl-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(l), C{2), C(3), and C(4), respectively, which shifted to 181.3,103.4,192.3,and29.9 ppm, respectively, when acetoacetyl-CoA was complexed with MCAD.While C(4) underwent a small upfield shift, the other carbons experienced significant shifts; both the C(l)andC(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 α-proton at C(2).This machinery is of fundamental importance in the initial step of MCAD catalysis. © by the Journal of Biochemistry Regular Papers nationallicence acetoacetyl-CoA nationallicence acyl-CoA dehydrogenase nationallicence 13C-NMR nationallicence flavoenzyme nationallicence nuclear magnetic resonance nationallicence Miura Retsu Departments of Biochemistry, Kumamoto University School of Medicine 2-2-1 Honjo, Kumamoto 960 aut Nishina Yasuzo Physiology, Kumamoto University School of Medicine 2-2-1 Honjo, Kumamoto 960 aut Fujii Shigeru Laboratory of Chemistry, Kansai Medical University Hirakata, Osaka 573 aut Shiga Kiyoshi Physiology, Kumamoto University School of Medicine 2-2-1 Honjo, Kumamoto 960 aut The Journal of Biochemistry Oxford University Press 119/3(1996-03), 512-519 0021-924X 119:3<512 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021271 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021271 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Miura Retsu Departments of Biochemistry, Kumamoto University School of Medicine 2-2-1 Honjo, Kumamoto 960 aut NATIONALLICENCE 700 1- Nishina Yasuzo Physiology, Kumamoto University School of Medicine 2-2-1 Honjo, Kumamoto 960 aut NATIONALLICENCE 700 1- Fujii Shigeru Laboratory of Chemistry, Kansai Medical University Hirakata, Osaka 573 aut NATIONALLICENCE 700 1- Shiga Kiyoshi Physiology, Kumamoto University School of Medicine 2-2-1 Honjo, Kumamoto 960 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/3(1996-03), 512-519 0021-924X 119:3<512 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford