Ring current effects in the active site of medium-chain Acyl-CoA dehydrogenase revealed by NMR spectroscopy.
Ring current effects in the active site of medium-chain Acyl-CoA dehydrogenase revealed by NMR spectroscopy.
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核磁共振波谱揭示中链酰基辅酶A脱氢酶活性位点的环电流效应。
DOI：
10.1021/ja050083p
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Tonge,PeterJ
中科院分区：
文献类型：
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作者：
Wu,Jiaquan;Bell,AlasdairF;Jaye,AndrewA;Tonge,PeterJ
中链酰基辅酶A脱氢酶（MCAD）催化黄素依赖性的脂肪酰基辅酶A氧化为相应的反式-2-烯酰基辅酶A。已使用13 C NMR和1H − 13 C HSQC光谱研究了产物类似物己二烯酰辅酶A（HD-CoA）与重组猪MCAD（pMCAD）的相互作用。在结合氧化的pMCAD后，C1、C2和C3 HD碳的化学位移分别向高场移动12.8、2.1和13.8 ppm。此外，C3-H的1H化学位移也向高场移动了1.31 ppm，而C4 HD-CoA碳的化学位移在结合后不变。考虑到先前的拉曼研究的结果，这些化学位移的变化是出乎意料的，所述拉曼研究揭示了C3 C2-C1 O HD烯酮片段在与MCAD结合时被极化，使得C3和C1碳上的电子密度降低，而不是增加（Pellet等人，Biochemistry 2000，39，13982 - 13992）。为了研究与MCAD结合的HD-CoA的NMR和拉曼数据之间的明显差异，已经获得了与烯酰-CoA水合酶结合的HD-CoA的13 C NMR光谱，所述烯酰-CoA水合酶是先前也使用拉曼光谱研究的酶系统。值得注意的是，与烯酰-CoA水合酶的结合导致C1和C3 HD碳的化学位移分别向低场移动4.8和5.6 ppm，而C2共振向高场移动2.2 ppm，这与从拉曼光谱预测的这些碳处的电子密度的改变非常一致（Bell，A. F.地;吴，J.;冯，Y.;通格，P.J.Biochemistry2001，40，1725 - 33）。在HD-CoA/MCAD复合物中C1和C3 HD碳所经历的屏蔽的大幅增加被提出是由黄素辅因子的异咯嗪部分的环电流场引起的。黄素环电流，这是唯一存在的酶被放置在一个外部磁场中，也解释了在13 C NMR化学位移的差异为乙酰乙酰辅酶A作为烯醇结合到MCAD和烯酰辅酶A水合酶，并用于合理化的观察，即C1和C3共振的线宽较窄时，配体被绑定到MCAD比当他们是免费的蛋白质溶液。
Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the flavin-dependent oxidation of fatty acyl-CoAs to the correspondingtrans-2-enoyl-CoAs. The interaction of hexadienoyl-CoA (HD-CoA), a product analogue, with recombinant pig MCAD (pMCAD) has been studied using13C NMR and1H−13C HSQC spectroscopy. Upon binding to oxidized pMCAD, the chemical shifts of the C1, C2, and C3 HD carbons are shifted upfield by 12.8, 2.1, and 13.8 ppm, respectively. In addition, the1H chemical shift of the C3-H is also shifted upfield by 1.31 ppm while the chemical shift of the C4 HD-CoA carbon is unchanged upon binding. These changes in chemical shift are unexpected given the results of previous Raman studies which revealed that the C3C2−C1O HD enone fragment is polarized upon binding to MCAD such that the electron density at the C3 and C1 carbons is reduced, not increased (Pellet et al.Biochemistry2000,39, 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, binding to enoyl-CoA hydratase causes the chemical shifts of the C1 and C3 HD carbons to move downfield by 4.8 and 5.6 ppm, respectively, while the C2 resonance moves upfield by 2.2 ppm, in close agreement with the alterations in electron density at these carbons predicted from Raman spectroscopy (Bell, A. F.; Wu, J.; Feng, Y.; Tonge, P. J.Biochemistry2001,40, 1725−33). The large increase in shielding experienced by the C1 and C3 HD carbons in the HD-CoA/MCAD complex is proposed to arise from the ring current field from the isoalloxazine portion of the flavin cofactor. The flavin ring current, which is only present when the enzyme is placed in an external magnetic field, also explains the differences in13C NMR chemical shifts for acetoacetyl-CoA when bound as an enolate to MCAD and enoyl-CoA hydratase and is used to rationalize the observation that the line widths of the C1 and C3 resonances are narrower when the ligands are bound to MCAD than when they are free in the protein solution.