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

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：化学键的形成和裂解在平面中间体的硅面或重面发生立体特异性反应，或者在两个面都发生非立体特异性反应。各种PLP酶的立体特异性已经研究了在其转氨化反应（包括副反应转氨化）中辅因子C-4′和底物部分之间的氢转移。立体特异性反映了酶的活性位点结构，特别是辅酶-底物希夫碱和氢转移催化碱的地形位置。目前所研究的PLP酶仅催化硅面特异性氢转移。这表明这些PLP酶具有相似的活性位点结构，并且更多的是从共同的祖先蛋白质进化而来的。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 -氨基酸转氨酶在中间体上催化表面氢转移，它们具有显著的序列同源性。d -氨基酸转氨酶的x射线结晶研究表明，该酶的催化碱基与辅助因子C4′的相对排列与催化硅面中间体的其他转氨酶相反。d -氨基酸转氨酶的折叠方式与目前已知的其他转氨酶不同。因此，基于一级结构、三维结构和氢转移立体化学的转氨酶分类是一致的。我们还发现，依赖plp的氨基酸消旋酶，其初级结构与其他plp酶不同，在平面中间体的两面催化非立体特异性氢转移。plp依赖性氨基酸外消旋酶可能是由与转氨酶不同的原始蛋白进化而来的。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