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Expression Mechanism of a Specific Catalytic Function in Flavoenzymes

黄素酶特定催化功能的表达机制

基本信息

批准号：
03680178
负责人：
FUJII Shigeru
金额：
$ 0.32万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03680178/
关键词：
Flavoenzyme Reaction Mechanism Electron Transfer NMR Resonance Raman Stable Isotope Labeling

项目摘要

The results of this research project were summarized as follows.(1) NAADPH-adrenodoxin reductase^<13>C-NMR spectra of this enzyme were observed by using ^<13>C-labeled FAD. In the complex of the enzyme with NADP, the signals for 4- and 10a-^<13>C shifted to up-fields by 2.1 and 0.7 ppm, respectively, and the 4a-^<13>C signal shifted to a down-field by 1.4 ppm in comparison with those of the free enzyme. These results suggest that the in the complex the pyridine moietly of NADP is located in the vicinity of C(4a)-C(4) region and that the pi-electron density of the 4a-position of FAD is decreased in the enzyme-NADP complex. This argues in favor of the electron transfer from NADPH to FAD of the enzyme.(2) old yellow enzymeThe enzyme was reconstituted with an active site probe, 8-fluoro-8-demethyl FMN. When the reconstituted enzyme was subjected to specific limited proteolysis with bovine alpha-chymotrypsin, it was separated to 14K and 34k fragments and then the coenzyme, FMN, was covalent … More ly bound to the N-terminal leucine of the 14K fragment. These results suggest that the 14K domain is the flavin-binding domain and the coenzyme is located near the N-terminal region of the 14K domain.(3) medium-chain acyl-CoA dehydrogenaseThis enzyme was studied by ^<13>C-and ^<15>N- NMR, and resonance Raman spectroscopies. In the ^<13>C- and ^<15>N-NMR spectra of the complex of the enzyme with acetoacetyl-CoA, 5-^<15>N resonance of FAD was specifically upfield-shifted from that of the free enzyme, while other resonances were not appreciably shifted. This suggests that acetoacetyl-CoA induces specific change in the electronic state at N(5) of FAD. Resonance Raman spectra of the complex of the enzyme with acetoacetyl-CoA and of the purple complex formed upon the addition of octanoyl-CoA to the enzyme showed that in the ligands of the complexes a large contribution of ionie resonance structures was C(2)=C(1)-O. This fact probably facilitates the abstraction of the alpha-proton of substrates. Less

主要研究结果如下：(1)用~(13)&lt；~(13)&gt；C标记的FAD对该酶的NAADPH-肾上腺素还原酶进行了核磁共振波谱观察。在酶与NADP的络合物中，与游离酶相比，4-和10a-β-lt；13&gt；C的信号分别上移了2.1和0.7ppm，4a-^&lt；13&gt；C的信号下移了1.4ppm。这些结果表明，在酶-NADP络合物中，NADP的吡啶主要位于C(4a)-C(4)区域附近，FAD的4a位的pi电子密度在酶-NADP络合物中降低。这有利于酶的电子从NADPH到FAD的转移。(2)老黄酶该酶是用活性中心探针8-氟-8-去甲基FMN重组的。重组酶用牛α-胰凝乳酶进行特异性限制性蛋白水解酶，将其分离为14K和34K片段，辅酶FmN为共价…。更多地与14K片段的N-末端亮氨酸结合。这些结果表明，14K结构域是黄素结合区，辅酶位于14K结构域的N-末端附近。(3)中链酰辅酶A脱氢酶用核磁共振和共振拉曼光谱研究了该酶。在酶与乙酰乙酰-CoA形成的络合物的^&lt；13&gt；C-和^&lt；15&gt；N-核磁共振谱中，FAD的5-^&lt；15&gt；N与游离酶的5-^&lt；15&gt；N的共振峰发生了明显的前移，而其他共振峰没有明显的位移。这表明乙酰乙酰-辅酶A在FAD的N(5)处诱导了特定的电子态变化。酶与乙酰乙酰-辅酶A形成的络合物和与辛酰辅酶A形成的紫色络合物的共振拉曼光谱表明，在络合物的配体中，离子共振结构的贡献很大，为C(2)=C(1)-O。这一事实可能有助于提取底物中的α-质子。较少