Ring Current Effects in the Active Site of Medium-Chain Acyl-CoA Dehydrogenase Revealed by NMR Spectroscopy

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• 作者：Jiaquan Wu ; Alasdair F. Bell ; Andrew A. Jaye ; and Peter J. Tonge
• 刊名：Journal of the American Chemical Society
• 出版年：2005
• 出版时间：June 15, 2005
• 年：2005
• 卷：127
• 期：23
• 页码：8424 - 8432
• 全文大小：197K
• 年卷期：v.127,no.23(June 15, 2005)
• ISSN：1520-5126

文摘

Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the flavin-dependent oxidation offatty acyl-CoAs to the corresponding trans-2-enoyl-CoAs. The interaction of hexadienoyl-CoA (HD-CoA),a product analogue, with recombinant pig MCAD (pMCAD) has been studied using ¹³C NMR and ¹H-¹³CHSQC spectroscopy. Upon binding to oxidized pMCAD, the chemical shifts of the C1, C2, and C3 HDcarbons are shifted upfield by 12.8, 2.1, and 13.8 ppm, respectively. In addition, the ¹H chemical shift ofthe C3-H is also shifted upfield by 1.31 ppm while the chemical shift of the C4 HD-CoA carbon is unchangedupon binding. These changes in chemical shift are unexpected given the results of previous Raman studieswhich revealed that the C3=C2-C1=O HD enone fragment is polarized upon binding to MCAD such thatthe electron density at the C3 and C1 carbons is reduced, not increased (Pellet et al. Biochemistry 2000,39, 13982-13992). To investigate the apparent discrepancy between the NMR and Raman data for HD-CoA bound to MCAD, ¹³C NMR spectra have been obtained for HD-CoA bound to enoyl-CoA hydratase,an enzyme system that has also previously been studied using Raman spectroscopy. Significantly, bindingto enoyl-CoA hydratase causes the chemical shifts of the C1 and C3 HD carbons to move downfield by4.8 and 5.6 ppm, respectively, while the C2 resonance moves upfield by 2.2 ppm, in close agreement withthe alterations in electron density at these carbons predicted from Raman spectroscopy (Bell, A. F.; Wu,J.; Feng, Y.; Tonge, P. J. Biochemistry 2001, 40, 1725-33). The large increase in shielding experiencedby the C1 and C3 HD carbons in the HD-CoA/MCAD complex is proposed to arise from the ring currentfield from the isoalloxazine portion of the flavin cofactor. The flavin ring current, which is only present whenthe enzyme is placed in an external magnetic field, also explains the differences in ¹³C NMR chemicalshifts for acetoacetyl-CoA when bound as an enolate to MCAD and enoyl-CoA hydratase and is used torationalize the observation that the line widths of the C1 and C3 resonances are narrower when the ligandsare bound to MCAD than when they are free in the protein solution.