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Studies on Expression Mechanism of a Specific Catalytic Function in Flavoenzymes

黄素酶特定催化功能的表达机制研究

基本信息

批准号：
09680634
负责人：
FUJII Shigeru
金额：
$ 0.7万
依托单位：
Kansai Medical Universtiy
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680634/
关键词：
Flavoenzyme Reaction Mechanism Electron Transfer NMR Stable Isotope Labeling ワラビン酵素

项目摘要

The results of this research project were summarized as follows. It was confirmed that old yellow enzyme (OYE) exists in cytosol of brewer's yeast cell, In the fermentation process using the brewer's yeast, the amount of OYE rapidly increased during the first 12 hrs of the fermentation and then gradually decreased in a low oxygen concentration. These results suggest that OYE works in the presence of oxygen. Old Yellow enzyme (OYB) from brewer's yeast was reconstituted with ^<13>C-enriched FMNs and 8-fluoro-8-demethyl-FMN (8F-FMN). Interactions of the reconstituted OYEs with NADP^+ and with cytochrome-c were investigated by ^<13>C- and ^<19>F-NMR spectroscopy. By the complex forma- tion between OYE and NADP^4, ^<19>F-NMR signal of 8F-FMN-OYE shifted tohigher field, and remarkable changes of 4a- and 10a-^<13>C NMR signalswere observed. When cytochrome c was bound to the reconstituted enzymes, 8-^<19>F and ^<23>C NMR signals shifted to higher fields, whereas 4-, 4a-, 10a-^<13>C NMR signals shifted to lower fields. It is tempting to speculate, on the basis of these findings, that the electron transfer from aYE to cytochrome c occurs through the xylene edge of FMNsubsequent to the electron density elevation at the xylene portion upon complex formation with cytochrome c. The expression system of acyl-CoA dehydrogenase has been established to yield an abundant enzyme. By use of this system, we will get more detailed information on the reaction mechanism of the enzyme.

该研究项目的结果总结如下。结果表明，啤酒酵母细胞胞浆中存在老黄酶（OYE），在啤酒酵母发酵过程中，OYE含量在发酵的前12小时内迅速增加，然后在低氧条件下逐渐减少。这些结果表明OYE在氧气存在下起作用。将来自啤酒酵母的老黄酶（OYB）用富含N2 C的<13>FMN和8-氟-8-去甲基-FMN（8 F-FMN）重构。用~ 13 C和~ 13 F-NMR光谱研究了重组OYE与NADP^+和细胞色素c的相互作用<13><19>。OYE与NADP形成络合物后，8 <19>F-FMN-OYE的~ 13 F-NMR信号向高场移动，4 α-和10 α-~ <13>13 CNMR信号发生显著变化。当细胞色素c与重组酶结合时，8- 13 <19>F和13 <23>C NMR信号向高场移动，而4-、4a-、10a-13 <13>C NMR信号向低场移动。这是诱人的推测，在这些研究结果的基础上，从aYE到细胞色素c的电子转移发生通过二甲苯边缘的FMN随后的电子密度升高后，在二甲苯部分与细胞色素c形成复合物。已建立了酰基辅酶A脱氢酶的表达系统，以产生丰富的酶。通过该系统的应用，我们将对酶的反应机理有更详细的了解。