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Elucidation of mechanism for catalytic action of pyridoxal enzymes

阐明吡哆醛酶的催化作用机制

基本信息

批准号：
07457031
负责人：
KAGAMIYAMA Hiroyuki
金额：
$ 4.86万
依托单位：
Osaka Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

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

吡哆醛酶的底物识别通过对天冬氨酸氨基转移酶（AST）、芳香族氨基酸氨基转移酶（ArT）和支链氨基酸氨基转移酶（BCT）在底物类似物存在下的X-射线晶体学分析，证明了它们的底物识别模式，在AST和ArT中，二羧酸底物的α和末端羧酸基团分别与Arg 386和Arg 292形成分叉氢键。而BCT的α-羧基识别不同：Tyr 95的OH和Gly 256-Ala 259的β-转角的两个肽NH基团。ArT通过重排活性位点残基侧链引起的氢键网络，将底物的酸性和芳香族侧链容纳在同一口袋中。利用DNA改组技术对AST基因进行随机突变，得到对支链氨基酸具有底物特异性的AST。吡哆醛酶的催化机理 ...更多信息对于AST和天冬氨酸盐的缔合，一种起始于未质子化的醛亚胺和单阴离子天冬氨酸盐，另一种起始于质子化的醛亚胺和双阴离子天冬氨酸盐。芳香族氨基酸脱羧酶（AADC）的Lys 303被发现是形成内部醛亚胺的残基。研究[Lys 303 Ala]酶的副反应，我们可以表明赖氨酸残基对于脱羧步骤不是必需的，但是对于产物释放是重要的。由底物结合引入的AADC的假定柔性区域的构象变化被证明对于外部醛胺形成是重要的，主要是通过分析在柔性区域内的键处裂解的片段酶，色氨酸酶在碱性pH下具有催化活性，尽管内部醛亚胺的非活性的醛胺结构是主要种类。我们可以揭示，在底物结合时，阿达明结构转化为活性酮烯胺结构。少