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

依赖于吡哆醛5‘-磷酸(PLP)的酶的反应通过底物和辅酶的阴离子Schiff碱中间体的形成进行。酶反应有三种立体化学可能性：键的形成和断裂是立体地发生在平面中间体的单面或表面上，或者是在两面上非立体专一性地发生。研究了各种PLP酶在转氨基反应中发生的辅因子C-4‘和底物部分之间的氢转移反应的立体专一性，包括副反应转氨基。立体专一性反映了酶的活性中心结构，特别是辅酶-底物Schiff碱和催化氢转移碱的形貌情况。到目前为止所研究的PLP酶只催化表面特异性的氢转移。这表明这些plp酶具有相似的活性中心结构和…。更多的是从一种共同的祖先蛋白分化进化而来的。我们最近建立了一种新的测定氢转移立体特异性的方法，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，它们催化了中间体上的表面氢转移。D-氨基酸氨基转移酶的X-射线结晶研究表明，该酶的催化碱基相对于辅因子的C4‘的相对排列与其他催化表面中间体的氨基转移酶相反。D-氨基酸转氨酶的折叠结构不同于已发现的其他转氨酶。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是一致的。我们还发现，依赖于PLP的氨基酸外消旋酶，其一级结构不同于其他PLP-酶，催化平面中间体两面的非立体特异性氢转移。依赖于PLP的氨基酸消旋酶可能是由不同于转氨酶的逆转录蛋白进化而来的。较少

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