Miura R, Miyake Y
Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka.
J Biochem. 1987 Dec;102(6):1345-54. doi: 10.1093/oxfordjournals.jbchem.a122180.
The 13C-NMR spectra of the reaction intermediates of D-amino acid oxidase (DAO) were measured with DAO reconstituted with FAD in which the 2-, 4-, 4a-, and 10a-positions of the isoalloxazine moiety were selectively 13C-enriched. The reaction intermediates used include charge-transfer complexes of the oxidized DAO with substrate intermediates and those of the reduced enzyme with substrate intermediates. For the former type of complex, the reaction intermediates with beta-cyano-D-alanine (D-BCNA) and D-proline were used, while for the latter the purple intermediates with D-alanine and D-proline were chosen. The 13C-resonances of 2-13C in the reaction intermediates with D-BCNA and D-proline were downfield-shifted by about 1 ppm relative to the free oxidized DAO. The 4-13C signal for the DAO-D-BCNA intermediate was observed at 1.2 ppm upfield from that of the oxidized DAO, though that for DAO-D-proline intermediate showed no shift. These results suggest modulation of the hydrogen bondings at C(2) = 0 and/or C(4) = 0 in these reaction intermediates. Comparison of the 13C-resonances of reduced DAO with those of free reduced FMN in the neutral and anionic forms indicate that FAD in reduced DAO is in the anionic reduced form. The 4a-13C resonance of reduced DAO is upfield-shifted by about 3 ppm from that of free reduced anionic FMN. Comparison of the 13C-resonances for the purple intermediates with those of reduced FMN and reduced DAO indicate unequivocally that FAD in the purple intermediate is in the anionic reduced state. The 4a-13C resonances for the purple intermediates were substantially upfield-shifted (by 2.4 ppm with D-alanine and 1.9 ppm with D-proline) relative to reduced DAO. This indicates that the electron density, and hence the nucleophilicity, of the 4a-carbon is elevated in the purple intermediate relative to free reduced DAO. This leads to a model in which the oxidative half reaction proceeds via the reaction of molecular oxygen at the 4a-position of the reduced FAD in the purple intermediate. This provides a rational molecular basis for the oxidative half reaction by way of the purple intermediate prior to product release rather than by way of free reduced enzyme after product release.
用在异咯嗪部分的2 -、4 -、4a -和10a -位选择性地用13C富集的FAD重组的D -氨基酸氧化酶（DAO）测量反应中间体的13C - NMR光谱。所使用的反应中间体包括氧化型DAO与底物中间体的电荷转移复合物以及还原型酶与底物中间体的电荷转移复合物。对于前一种类型的复合物，使用了与β -氰基 - D -丙氨酸（D - BCNA）和D -脯氨酸的反应中间体，而对于后一种类型，选择了与D -丙氨酸和D -脯氨酸的紫色中间体。与游离氧化型DAO相比，与D - BCNA和D -脯氨酸的反应中间体中2 - 13C的13C共振向低场移动了约1 ppm。DAO - D - BCNA中间体的4 - 13C信号在比氧化型DAO的信号高场1.2 ppm处观察到，而DAO - D -脯氨酸中间体的该信号没有位移。这些结果表明这些反应中间体中C(2)=O和/或C(4)=O处的氢键发生了调制。中性和阴离子形式的还原型DAO的13C共振与游离还原型FMN的13C共振的比较表明，还原型DAO中的FAD处于阴离子还原形式。还原型DAO的4a - 13C共振比游离还原型阴离子FMN的共振向高场移动了约3 ppm。紫色中间体的13C共振与还原型FMN和还原型DAO的13C共振的比较明确表明，紫色中间体中的FAD处于阴离子还原状态。紫色中间体的4a - 13C共振相对于还原型DAO大幅向高场移动（与D -丙氨酸反应时移动2.4 ppm，与D -脯氨酸反应时移动1.9 ppm）。这表明紫色中间体中4a -碳的电子密度以及因此的亲核性相对于游离还原型DAO有所提高。这导致了一个模型，其中氧化半反应通过紫色中间体中还原型FAD的4a -位处的分子氧反应进行。这为在产物释放之前通过紫色中间体而不是在产物释放之后通过游离还原型酶进行氧化半反应提供了合理的分子基础。
