Elucidation of mechanism for catalytic action of pyridoxal enzymes

吡哆醛酶的底物识别在底物类似物存在的情况下，对天冬氨酸转氨酶（AST）、芳香氨基酸转氨酶（ArT）和支链氨基酸转氨酶（BCT）进行x射线晶体分析，验证了它们的底物识别模式。在AST和ArT中，二羧基底物的α和远端羧酸基分别与Arg386和Arg292形成分叉氢键。然而，BCT的α -羧酸基识别是不同的：Tyr95的OH和由Gly256-Ala259组成的β -turn的两个肽NH基团。ArT通过重新排列一些活性位点残基侧链引起的氢键网络，将底物的酸性和芳香侧链容纳在同一个口袋中。通过DNA洗牌随机突变AST基因，获得对支链氨基酸具有底物特异性的新AST。吡哆醛酶与天冬氨酸缔合的两种途径可以从动力学上分离出来，一种是未质子化的醛胺与单阴离子天冬氨酸缔合，另一种是质子化的醛胺与双阴离子天冬氨酸缔合。早先的建议没有考虑到后者。发现芳香氨基酸脱羧酶（AADC）的Lys303是形成内部醛胺的残基。通过分析[Lys303 Ala]酶的副反应，我们可以发现赖氨酸残基在脱羧过程中不是必需的，但对产物的释放很重要。底物结合引起的AADC柔性区域的构象变化对外部醛胺的形成很重要，主要是通过分析柔性区域内键处的酶裂解片段。色氨酸酶在碱性条件下具有催化活性，尽管内部醛胺结构失活。我们可以发现，醛胺结构在底物结合后转化为活性酮胺结构。少

Substrate Recogntion of Pyridoxal EnzymesX-ray crystallographic analyzes of aspartate aminotransferase (AST), aromatic amino acid aminotransferase (ArT) and branched-chain amino acid aminotransferase (BCT), in the presence of substrate analogue, domonstrated the mode for their substrate recognition.In AST and ArT,the alpha and distal carboxylate groups of a dicarboxylic substrate form bifurcated hydrogen bonds with Arg386 and Arg292, respectively. However, the alpha-carboxylate group recognition of BCT was different : OH of Tyr95 and two peptide NH groups of beta-turn comprising Gly256-Ala259.ArT accomodates the acidic and aromatic side chains of substrates at the same pocket by rearranging the hydrogen bond networks caused by reorientation of the side chains of some active site residues. Random mutation of AST gene by DNA shuffling gave new AST with substrate specificity for branched-chain amino acid.Catalytic mechanism of Pyridoxal EnzymesWe could separate kinetically the two routes … More for association of AST and asparate, the one begins with the unprotonated aldimine and monoanionic aspartate, and the other with the protonated aldimine and dianionic aspartate. The earlier proposals have not considered the latter.Lys303 of aromatic amino acid decarboxylase (AADC) was found to be the residue forming the internal aldimine. Examining the side reaction of [Lys303 Ala] enzyme, we could indicate that the lysine residue is not essential for the decarboxylation step, but important for the product release.The conformational change of the putative flexible region of AADC introduced by the substrate binding was shown to be important for the external aldmine formation, mainly by analyzing the fragmentary enzyme cleaved at the bond inside the flexible region.Tryptophanase is catalytically activ in alkaline pH in spite of inactive aldamine structure of the internal aldimine to be the major species. We could reveal that the aldamine structure was converted to the active ketoenamine structure upon the substrate-binding. Less