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

吡哆醛 5'-磷酸 (PLP) 依赖性酶的反应通过底物和辅酶的阴离子希夫碱中间体的形成进行，酶反应存在三种立体化学可能性：键的形成和断裂在任一者上立体特异性地发生。平面中间体的 si 或重新面，或者在两个面上进行非立体定向已经研究了各种 PLP 酶的 C-4' 之间的氢转移的立体定向性。立体特异性反映了酶的活性位点结构，尤其是辅酶底物席夫碱和氢转移催化碱的拓扑情况。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，并且与其他酶不同。我们最近建立了一种测定氢转移立体特异性的新方法，发现D-氨基酸转氨酶和支链L-氨基酸转氨酶表现出显着的序列同源性，催化reface氢转移。 D-氨基酸转氨酶的 X 射线晶体学研究揭示了该酶催化碱基与辅因子 C4' 的相对排列。与催化si-face中间体的其他转氨酶的折叠方式相反，D-氨基酸转氨酶的折叠与迄今为止所证明的其他转氨酶的折叠不同，因此，基于一级结构、三维结构的转氨酶的分类。我们还发现PLP依赖性氨基酸消旋酶的一级结构与其他PLP酶催化的不同。平面中间体两个面上的非立体特异性氢转移可能是从与氨基转移酶不同的祖先蛋白质进化而来的。