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Stereochemical Studies of the Structure, Function, and Molecular Evolution of Pyridoxal Enzymes

吡哆醛酶的结构、功能和分子进化的立体化学研究

基本信息

批准号：
06680611
负责人：
YOSHIMURA Tohru
金额：
$ 1.34万
依托单位：
KYOTO 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：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

The reactions of pyridoxal 5'-phosphate (PLP)-dependent enzymes proceed through the formation of an anionic Schiff base intermediate of the substrate and the coenzyme. Three stereochemical possibilities exist for the enzyme reactions : The formation and cleavage of bonds occur stereospecifically on either si-or re-face of the plannar intermediate, and alternatively non-stereospecifically on both faces. The stereospecificities of various PLP enzymes have been studied for the hydrogen transfer between C-4' of the cofactor and substrate moieties which occurs in their transamination reactions including a side-reaction transamination. The stereospecificities reflect the active-site structures of the enzymes, especially the topographical situation of a coenzyme-substrate Schiff base and a catalytic base for the hydrogen transfer. The PLP enzymes so far studied catalyze only the si-face specific hydrogen transfer. This suggests that these PLP enzymes have the similar active-site structure and … More are evolved divergently from a common ancestral protein. We recently established a new method for the determination of stereospecificity for the hydrogen transfer, and found that D-amino acid aminotransferase and branched chain L-amino acid aminotransferase, which show a significant sequence homology, catalyze the reface hydrogen transfer on the intermediate. The X-ray chrystallographical studies of D-amino acid aminotransferase revealed that the relative arrangement of the catalytic base of the enzyme to the C4' of the cofactor is opposite to that of other aminotransferases catalyzing the si-face intermediate. The fold of D-amino acid aminotransferase is different from those of other aminotransferase so far demonstrated. Therefore, the classifications of the aminotransferases based on the primary structure, three dimensional structure, and stereochemistry of the hydrogen transfer coincide with one another. We also found that PLP-dependent amino acid racemases, whose primary structures are different from those of other PLP-enzymes catalyze the non-stereospecific hydrogen transfer on both faces of the planar intermediate. The PLP-dependent amino acid racemases are probably evolved from the ancesrtral protein which differes to those of aminotransferases. Less

5 '-磷酸吡哆醛（PLP）依赖性酶的反应通过形成底物和辅酶的阴离子希夫碱中间体来进行。酶反应存在三种立体化学可能性：键的形成和裂解立体特异性地发生在平面中间体的Si-或Re-face上，或者非立体特异性地发生在两面上。已经研究了各种PLP酶的立体特异性，用于辅因子和底物部分的C-4'之间的氢转移，所述氢转移发生在它们的转氨反应（包括副反应转氨）中。立体专一性反映了酶的活性中心结构，特别是辅酶-底物席夫碱和氢转移催化碱的拓扑位置。迄今为止研究的PLP酶仅催化si-面特异性氢转移。这表明这些PLP酶具有相似的活性位点结构， ...更多信息都是从一个共同的祖先蛋白进化而来的。我们最近建立了一种新的测定氢转移立体专一性的方法，发现D-氨基酸氨基转移酶和支链L-氨基酸氨基转移酶具有显著的序列同源性，催化中间体上的重新表面氢转移。D-氨基酸氨基转移酶的X-射线化学异构研究表明，该酶的催化碱基与辅因子的C4'的相对排列与催化si-面中间体的其他氨基转移酶相反。D-氨基酸氨基转移酶的倍数不同于目前已证实的其他氨基转移酶。因此，基于氢转移的一级结构、三维结构和立体化学的氨基转移酶的分类彼此一致。我们还发现，PLP依赖的氨基酸消旋酶，其一级结构是不同于其他PLP-酶催化的平面中间体的两面上的非立体特异性氢转移。依赖PLP的氨基酸消旋酶可能是由类似于氨基转移酶的前体蛋白进化而来。少