Interactions in the Site of Acetylcholinesterase

乙酰胆碱酯酶位点的相互作用

• 批准号: 6899197
• 负责人: TERRONE L ROSENBERRY
• 金额: $ 26.1万
• 依托单位: MAYO CLINIC JACKSONVILLE
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6899197
• 关键词: acetylcholinesterase; active sites; conformation; crystallization; enzyme structure; fluorescent dye /probe; hydrolysis; phosphorylation; protein binding; protein protein interaction; riboflavin; site directed mutagenesis

DESCRIPTION (provided by applicant): Acetyicholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetyicholine. We will focus on molecular mechanisms responsible for the high catalytic efficiency of AChE. Such information may allow the design of new classes of drugs to protect against organophosphate toxicity. The structure of AChE reveals an active site gorge with two sites of ligand interaction separated by 12-15 A: an acylation site (or A site) at the base of the gorge, and a peripheral site (or P site) at its mouth. We discovered that substrates or inhibitors that bind to the P site of AChE impose a steric blockade on ligand entry to and exit from the A site. We now find that ligand binding to the P site also increases the acylation rate constant for a substrate bound to the A site, while binding of the neurotoxin fasciculin to the P site results in a dramatic decrease in these acylation rate constants. We will test a mechanistic model involving the P and A sites that can account for these features. Our first aim examines thioflavin T, whose fluorescence is enhanced when it is bound to the P site and partially quenched when ligands bind to the A site to form a ternary complex, indicating a local conformational change. We will pursue crystal structure determinations of the AChE-thioflavin T complex with collaborators and use thioflavin T binding and fluorescence to monitor acceleration of organophosphorylation rate constants with wild type and mutant AChEs. In a new second aim, we will modify the SH groups of mutants E81C and E84C with a solvent-sensitive fluorophore and examine interactions with the P and A sites. We will ask whether ligand binding to the P site alone is sufficient to induce conformational movement of the peptide loop that contains these residues. Our third aim is to determine whether substrates closely analogous to acetyicholine can accelerate acylation reactions by binding to the P site. We will test our kinetic model by comparing predicted and measured P site substrate affinities for acetylthiocholine and m-(trimethylammonio)acetanilide in wild type, W86F and W86A AChEs. Our fourth aim is to identify the AChE residues through which fasciculin exerts an inhibitory conformational effect on the A site. Our hypothesis, that a segment of an Omega-loop (residues 72 to 87) is involved in transducing this effect, is supported by data showing loss of much of the inhibitory effect with the D74G and W86F mutants. To test this hypothesis further, we will construct additional site-specific AChE mutants as well as fasciculin mutants and determine whether these residues can be altered to retain inhibition of organophosphorylation but decrease inhibition of substrate hydrolysis.

描述（由申请人提供）：乙甲乙烯酸酯酶（ACHE）通过水解神经递质乙酰氨基氨酸来终止胆碱能突触的突触传播。我们将专注于负责ACHE催化效率高的分子机制。这样的信息可能允许设计新的药物以防止有机磷酸盐的毒性。 ACHE的结构揭示了一个活跃的现场峡谷，其两个配体相互作用的位置由12-15 A分隔为12-15 A：峡谷底部的酰化位点（或一个位置），以及一个在其嘴里的外围部位（或P位点）。我们发现与ACHE P位点结合的底物或抑制剂在配体进入并从A位置退出时会施加空间阻滞。现在，我们发现与P位点的配体结合还增加了与A位点结合的底物的酰化速率常数，而神经毒素筋膜与P位点的结合导致这些酰基化速率常数急剧下降。我们将测试一个涉及P和A站点的机械模型，该模型可以解释这些功能。我们的第一个目的检查了硫非黄素T，当配体与A位点结合以形成三元络合物时，其荧光会增强，并部分淬火，表明局部构象变化。我们将与合作者一起追求ACHE-噻ti-黄酮T络合物的晶体结构，并使用硫氟硫蛋白T结合和荧光来监测具有野生型和突变酸痛的有机磷酸化速率常数的加速度。在新的第二个目标中，我们将用溶剂敏感的荧光团修改突变体E81C和E84C的SH组，并检查与P和A位点的相互作用。我们将询问配体与P位点的结合是否足以诱导包含这些残基的肽环的构象运动。我们的第三个目的是确定底物是否与乙酰氨酸密切相似，可以通过与P位点结合来加速酰基化反应。我们将通过比较乙酰噻吩胆碱和M-（三甲基氨基苯甲酸）乙酰苯胺中的预测和测量的P位点底物亲和力来测试我们的动力学模型。我们的第四个目的是确定筋膜蛋白对A位点产生抑制构象作用的ACHE残基。我们的假设是，欧米茄环（残基72至87）参与了转导这种效果，这表明数据显示了与D74G和W86F突变体失去大部分抑制作用的数据。为了进一步检验该假设，我们将构建其他位点特异性的酸痛突变体和筋膜突变体，并确定是否可以改变这些残基以保留有机磷酸化的抑制，但会降低底物水解的抑制作用。