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Studies on the Reaction Mechanisms of Flavoenzymes by Multi-Nuclear NMR

多核核磁共振研究黄素酶的反应机理

基本信息

批准号：
01480525
负责人：
MIURA Retsu
金额：
$ 4.1万
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 (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480525/
关键词：
Flavoenzyme Reaction Mechanism Electron Transfer Nuclear Magnetic Resonance NMR Multi-nuclear NMR Stable Isotope Label

项目摘要

1. D-AMINO ACID OXIDASE(DAO)Substrate analogs, o-, m-, and p-flucrobenzoate were used for elucidating the electron transfer from a substrate to oxidized flavin by means of F(19)NMR. Of the NMR signals of these analogs, the signal of o-derivative exhibited the largest chemical shift change when bound to DAO, while those ofm- and p-derivativesexhibitedmoderateandminorchanges, respectively. This is consistent with the electron transfer from the amino nitrogen of a substrate to the oxidized flavin.NADPH-ADRENODOXIN REDUCTASE(ADR)ADR was reconstituted with C(13)-enriched FAD and the C- 13 NMR spectra of the reconstituted ADR in the free state and in the complexed state with NADP were measured. The positions enriched were 2, 4, 4a, and loa-positions of the flavin nucleus. In the complexed form with NADP, the 4a-signal-was down-field shifted, suggesting that when electrons are transferred from NADPH to oxidized flavin the NADPH-binding lowers the electron density at the 4a-position thereby facilitating the electron transfer.3. OLD YELLOW ENZYME(OYE)When OYE reconstituted with 8-fluoro-FMN was subjected to limited proteolysis by chymotrypsin, the protein was specificaly cleaved to yield 14KD and 34KD fragments. Subsequently, the amino group of the leucine residue of the N-terTninus of the 14KD fragment generated by the cleavage attacked the 8-position of 8-fluoro-FMN nucleophilically and a covalent bond was fontied between the flavin moiety and the 14KD fragment. From these results we could deduce the position of FMN binding and the mode of binding of FMN in OYF-

1. D-氨基酸氧化酶（DAO）底物类似物邻、间和对氟苯甲酸酯用于通过F（19）NMR阐明从底物到氧化黄素的电子转移。在这些类似物的NMR信号中，邻位衍生物与DAO结合时的化学位移变化最大，而间位和对位衍生物的化学位移变化较小。用富含C（13）的FAD重组NADPH-ADRENODOXIN REDUCTASE（ADR）ADR，测定了重组ADR游离态和与NADP复合态的C- 13 NMR谱。富集部位为黄素核的2、4、4a和10 a位。在与NADP的复合形式中，4a-信号-被向下场移动，这表明当电子从NADPH转移到氧化黄素时，NADPH结合降低了4a-位置的电子密度，从而促进了电子转移。当用8-氟-FMN重构的OYE被胰凝乳蛋白酶进行有限的蛋白酶解时，该蛋白被特异性地切割，产生14 KD和34 KD片段。随后，切割产生的14 KD片段的N-末端亮氨酸残基的氨基亲核攻击8-氟-FMN的8-位，并在黄素部分和14 KD片段之间形成共价键。根据这些结果，我们可以推测FMN在OYF中的结合位置和结合方式。