Catalytic Mechanism of Acetoacetate Decarboxylase

乙酰乙酸脱羧酶的催化机制

• 批准号: 9630430
• 负责人: Karen Allen
• 金额: $ 31.4万
• 依托单位: Trustees of Boston University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-15 至 2000-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9630430&HistoricalAwards=false
• 关键词: Catalytic; Mechanism; Acetoacetate; Decarboxylase

9630430 Allen The formation of a Schiff base (imine) between a carbonyl group and an amine nucleophile, with loss of water, is a prevalent reaction whose importance as an intermediate in enzyme catalyzed reactions has become apparent in the past five decades. The long-term objective of the proposed research is to define the structural and chemical mechanisms utilized by enzymes to promote the formation and reactivity of Schiff-base intermediates. A combination of the complementary techniques of X-ray crystallography, protein chemistry and kinetics will be used as tools. The proposed model system is the enzyme acetoacetate decarboxylase (AADase) complexed with its substrates, products and covalent intermediates or their stable analogs. The enzyme catalyzes the decarboxylation of acetoacetate to acetone and carbon dioxide is a "simple" decarboxylase, requiring no cofactors, instead using the -amino group of enzymic Lysl 15 with a pKa_6.0 to form the reactive covalent adduct. The X-ray crystallographic structure of the native, uncomplexed AADase enzyme will be determined. The structures of several enzyme-ligand complexes will also be solved: AADase will be complexed with 1) 2-oxopropane sulfonate, a substrate analog, 2~ acetowruvate and acetoacetone, covalent intermediate analogs, and 3) acetone, the product. These structures will act as "snap-shots" along the catalytic pathway of the enzyme. Kinetic experiments to uncover the rate limiting step(s) will be performed. These will consist of the measurement of reaction rates with synthetic substrates and acylating agents bearing electron donating and withdrawing groups. Intermediate trapping and solvent exchange experiments will be performed on mutant K1 16R AADase (20% wild-type activity) in which the pKa perturbing Iysine 116 has been mutated to arginine to assess whether there has been a change in the rate-limiting step(s) and/or stability ofthe Schiffbase. The information afforded by these studies will allow us to assess the chemical and struct ural contributions of the enzyme to the stability and reactivity of the essential Schiff-base intermediate. This approach could ultimately be extended to the analysis of less well-defined enzyme systems. %%% Underlying the proposed research is the understanding of the basic methods used by enzymes to increase the rate of chemical reactions by as much as one billion fold. The proposed studies will dissect the chemical steps used by one enzyme, acetoacetate decarboxylase (AADase), to achieve such rate accelerations. The chemical steps will be examined in two ways 1) by comparing the velocity of the reaction of AADase with its substrate (the substance with which it normally interacts) and chemically altered substrates and 2) by measuring the velocity of reactions promoted by AADases in which the pieces of the enzyme which participate in the chemical reactions have been altered using molecular biology. These chemical studies will then be correlated with studies of the threedimensional structure of the enzyme in atomic detail. Using the method of X-ray crystallography, we can visualize the exact position of the atoms that make up the protein AADase, thus obtaining a detailed picture of the shape of the catalytic machinery. Such pictures will be determined for AADase, alone and in the presence of substances called inhibitors, which resemble substrate, but which cannot participate in chemical reactions with the enzyme. Also, the interaction of AADase with the end-product of the normal chemical reaction can be visualized by this technique. These structures will act as "snapshots" along the pathway of the enzyme. Understanding of the relationship between structure and J~nct~on for the enzyme AADase can ultimately be used to understand other enzymatic reactions as well as to design enzymes with novel functions. ***

9630430艾伦：席夫碱(亚胺)是一种普遍存在的反应，在过去的五十年里，作为酶催化反应的中间体，席夫碱(亚胺)在羰基和胺亲核试剂之间的形成是一种非常普遍的反应。拟议研究的长期目标是确定酶用于促进席夫碱中间体的形成和反应的结构和化学机制。X射线结晶学、蛋白质化学和动力学等互补技术的组合将被用作工具。所提出的模型体系是乙酰乙酸酯脱羧酶(AADase)与其底物、产物和共价中间体或它们的稳定类似物络合而成。该酶催化乙酰乙酸酯脱羧制丙酮，二氧化碳是一种简单的脱羧酶，不需要辅因子，而是利用酶Lys1 15的氨基与pKa_6.0形成反应性共价加合物。将测定天然的、不复杂的AADase的X射线晶体结构。几种酶-配体复合体的结构也将被解决：AADase将与1)底物类似物2-氧代丙磺酸盐，2-乙酰丙酮酸和乙酰丙酮，共价中间类似物，以及3)丙酮，产物络合。这些结构将在酶的催化途径上起到“快照”的作用。将进行动力学实验，以揭示限速步骤(S)。这将包括测量与合成底物和带有电子给体和引出基团的酰化剂的反应速率。对突变的K1 16R AADase(野生型活性20%)进行中间捕获和溶剂交换实验，其中PKA干扰赖氨酸116突变为精氨酸，以评估限速步骤(S)是否改变和/或Schiffbase的稳定性。这些研究提供的信息将使我们能够评估酶对基本席夫碱中间体的稳定性和反应活性的化学和结构贡献。这种方法最终可以扩展到对定义不太明确的酶系统的分析。这项拟议的研究的基础是对酶所使用的基本方法的理解，这些方法可以将化学反应速度提高多达10亿倍。这项拟议的研究将剖析一种酶--乙酰乙酸脱羧酶(AADase)--实现这种速度加速所使用的化学步骤。化学步骤将通过两种方式进行检验，1)通过比较AADase与其底物(它通常与之相互作用的物质)和化学改变的底物的反应速度，以及2)通过分子生物学测量参与化学反应的酶片段已经改变的AADase促进的反应速度。然后，这些化学研究将与酶的原子细节的三维结构研究相关联。利用X射线结晶学的方法，我们可以直观地看到组成蛋白质AADase的原子的确切位置，从而获得催化机械形状的详细图像。这样的图片将被确定为AADase，单独和存在的物质称为抑制物，类似于底物，但不能参与与酶的化学反应。此外，AADase与正常化学反应的最终产物的相互作用也可以通过这一技术可视化。这些结构将沿着酶的途径起到“快照”的作用。了解AADase的结构与Jnct~on之间的关系，最终可以用来理解其他酶反应以及设计具有新功能的酶。***