• 主题
高级搜索  >
历史搜索 清空历史
首页> 美国卫生研究院文献>Plant Physiology >Mutagenic Definition of a Papain-Like Catalytic Triad Sufficiency of the N-Terminal Domain for Single-Site Core Catalytic Enzyme Acylation and C-Terminal Domain for Augmentative Metal Activation of a Eukaryotic Phytochelatin Synthase
【2h】

Mutagenic Definition of a Papain-Like Catalytic Triad Sufficiency of the N-Terminal Domain for Single-Site Core Catalytic Enzyme Acylation and C-Terminal Domain for Augmentative Metal Activation of a Eukaryotic Phytochelatin Synthase

机译：木瓜蛋白酶催化三联体的诱变定义单站点核心催化酶酰化作用的N末端结构域的充分性和真核植物螯合酶合金属的C末端结构域的充分性。

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务，本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文，但由于OA文献来源多样且变更频繁，仍可能出现获取不到、文献不完整或与标题不符等情况，如果获取不到我们将提供退款服务。请知悉。
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Phytochelatin (PC) synthases are γ-glutamylcysteine (γ-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (γ-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes γ-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes γ-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent γ-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary γ-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary γ-Glu-Cys acylation.
机译：植物螯合素（PC）合成酶是γ-谷氨酰半胱氨酸（γ-Glu-Cys）二肽基转肽酶，可催化由谷胱甘肽（GSH）和/或短链PC合成重金属结合的PC，（γ-Glu-Cys）nGly聚合物。 。通过对拟南芥（Arabidopsis thaliana; AtPCS1）酶的研究表明，尽管仅蛋白质的N端一半就足以通过形成单点酶酰基中间体来进行核心催化，但这是对于第二个位点的酰化和游离Cd 2 + 的增强刺激作用还不够。纯化的仅标记酶的前221个N端氨基酸残基（HIS-AtPCS1_221tr）的带有N-端六组氨酸标签的AtPCS1截短物可在含有Cd 2 + 2 + 过程中在两个位点经历了γ-Glu-Cys酰化反应，并受到游离态的刺激当从S-甲基谷胱甘肽合成S-甲基-PCs时，Cd 2 + ，当GSH是底物且不被直接刺激而是被抑制时，HIS-AtPCS1_221tr仅在一个位点发生γ-Glu-Cys酰化。 ，当S-甲基谷胱甘肽为底物时，游离Cd 2 + 。通过应用能够检测远距离同源性的序列搜索算法，我们在之前进行了简短报道，但没有完整报道，已确定AtPCS1的N端一半及其与其他来源的等价物具有类似木瓜蛋白酶的特征。 ，Clan CA Cys蛋白酶。从这些分析中得出的折叠分配可以证实并通过最近确定的来自蓝细菌Nostoc的远距离原核PC合酶同系物的晶体结构得以证实，这可以解释对保守的Cys残基Cys-56的严格要求就AtPCS1而言，为了形成具有生物合成能力的γ-Glu-Cys酶酰基中间体，来自实验的主要数据旨在确定推定的木瓜蛋白酶样催化三联体的其他两个残基His-162和Asp-180对AtPCS1的催化作用至关重要，但尚未提出。系统定点诱变研究的结果解决了我们对AtPCS1的基本理解的不足，该研究表明，净PC合成确实不仅需要Cys-56，而且还需要His-162和Asp-180。因此，通过实验确定了AtPCS1和其他真核PC合成酶是木瓜蛋白酶Cys蛋白酶超家族成员，但不同于它们的原核对等成员，它们除了具有木瓜蛋白酶样的N末端催化结构域外，还经历了初级γ- Glu-Cys酰化包含一个辅助金属感应的C末端结构域，该结构会进行二次γ-Glu-Cys酰化。

著录项

相似文献

  • 外文文献
  • 中文文献
代理获取

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号