Probing hydrogen-bonding interactions in the active site of medium-chain acyl-CoA dehydrogenase using Raman spectroscopy.
Probing hydrogen-bonding interactions in the active site of medium-chain acyl-CoA dehydrogenase using Raman spectroscopy.
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使用拉曼光谱探测中链酰基辅酶 A 脱氢酶活性位点的氢键相互作用。
DOI：
10.1021/bi0344578
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发表时间：
2003
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;Luo,Lian;Stephens,AveryW;Stankovich,MarianT;Tonge,PeterJ
用2‘-脱氧-FAD重组酶研究了氧阴离子空穴在猪中链酰辅酶A脱氢酶(PMCAD)催化反应中的作用。重组表达的WT pMCAD氧化正辛酰辅酶A(C8-CoA)的kcat(18.8±0.5)S~(-1)和Km(2.5±0.4μM)与从猪肾分离的天然pMCAD相似。与以前的研究一致[Engst等人]。(1999年)BioChemical 38,257−267]，WT酶与2‘-脱氧-FAD重组可使墨水量大大减少(400倍)，但对Km影响不大。为了研究底物与酶的氧阴离子空穴之间氢键的变化导致活性变化的分子基础，用拉曼光谱研究了与2‘-脱氧-FAD重组酶结合的产物类似物己二烯基辅酶A(HD-CoA)的结构。重要的是，当WT pMCAD使HD-CoA与2‘-脱氧-Fad pMCAD结合时，HD-CoA的Enone带的振动频率从1595下降到1568 cm-1，而enone带只移动了10 cm-1。因此，去除2‘-核糖基羟基会导致酶极化ES络合物基态的能力大大降低。根据对类似体系的分析，估计基态失稳降低了17kJ·mol-1，而反应活化能提高了15kJ·mol-1。此外，去除2‘-核糖基羟基使HD-CoA结合引起的氧化还原电位从18kJ·mol-1降低到11kJ·mol-1。因此，虽然氧阴离子空穴中氢键引起的配体极化与底物周转密切相关，但其他因素肯定是配体诱导的氧化还原电位变化的原因。最后，用谷氨酰胺取代催化碱基E376取消了酶催化底物氧化和催化C8-CoAα-质子与溶剂氢交换的能力，但2‘-脱氧-FAD重组酶催化α-质子交换的速率(Kexc)为0.085 S-1，仅比wt pMCAD(0.35 S-1)慢4倍。因此，与其在底物氧化中的作用相比，氧阴离子空穴在稳定α-质子交换过渡态方面只起到很小的作用，或者Kexfor WT pMCAD的值反映了E376COOH质子与溶剂交换的过程。
The role of the oxyanion hole in the reaction catalyzed by pig medium-chain acyl-CoA dehydrogenase (pMCAD) has been investigated using enzyme reconstituted with 2‘-deoxy-FAD. Thekcat(18.8 ± 0.5 s-1) andKm(2.5 ± 0.4 μM) values for the oxidation ofn-octanoyl-CoA (C8-CoA) by WT pMCAD recombinantly expressed inEscherichia coliare similar to those of native pMCAD isolated from pig kidney. In agreement with previous studies [Engst et al. (1999)Biochemistry 38, 257−267], reconstitution of the WT enzyme with 2‘-deoxy-FAD causes a large (400-fold) decrease inkcatbut has little effect onKm. To investigate the molecular basis for the alterations in activity resulting from changes in hydrogen bonding between the substrate and the enzyme's oxyanion hole, the structure of the product analogue hexadienoyl-CoA (HD-CoA) bound to the 2‘-deoxy-FAD-reconstituted enzyme has been probed by Raman spectroscopy. Importantly, while WT pMCAD causes a 27 cm-1decrease in the vibrational frequency of the HD enone band, from 1595 to 1568 cm-1, the enone band is only shifted 10 cm-1upon binding HD-CoA to 2‘-deoxy-FAD pMCAD. Thus, removal of the 2‘-ribityl hydroxyl group results in a substantial reduction in the ability of the enzyme to polarize the ground state of the ES complex. On the basis of an analysis of a similar system, it is estimated that ground state destabilization is reduced by up to 17 kJ mol-1, while the activation energy for the reaction is raised 15 kJ mol-1. In addition, removal of the 2‘-ribityl hydroxyl reduces the redox potential shift that is induced by HD-CoA binding from 18 to 11 kJ mol-1. Consequently, while ligand polarization caused by hydrogen bonding in the oxyanion hole is intimately linked to substrate turnover, additional factors must be responsible for ligand-induced changes in redox potential. Finally, while replacement of the catalytic base E376 with Gln abolishes the ability of the enzyme to catalyze substrate oxidation and to catalyze the exchange of the C8-CoA α-protons with solvent deuterium, the 2‘-deoxy-FAD-reconstituted enzyme catalyzes α-proton exchange at a rate (kexc) of 0.085 s-1, which is only 4-fold slower thankexcfor WT pMCAD (0.35 s-1). Thus, either the oxyanion hole plays only a minor role in stabilizing the transition state for α-proton exchange, in contrast to its role in substrate oxidation, or the value ofkexcfor WT pMCAD reflects a process such as exchange of the E376 COOH proton with solvent.