Catalytic Mechanism of Acetoacetate Decarboxylase

9630430艾伦在羰基和胺亲核试剂之间形成席夫碱（亚胺）并损失水是一种普遍的反应，其作为酶催化反应中的中间体的重要性在过去五十年中变得显而易见。这项研究的长期目标是确定酶促进席夫碱中间体形成和反应的结构和化学机制。X射线晶体学，蛋白质化学和动力学的互补技术的组合将被用作工具。所提出的模型系统是酶乙酰乙酸脱羧酶（AAD酶）与其底物，产物和共价中间体或其稳定的类似物复合。该酶催化乙酰乙酸脱羧为丙酮和二氧化碳，是“简单”脱羧酶，不需要辅因子，而是使用pKa_6.0的酶Lys 115的-氨基形成反应性共价加合物。将测定天然未复合AAD酶的X射线晶体结构。几种酶-配体复合物的结构也将得到解决：AAD酶将与1）2-氧代丙烷磺酸盐（底物类似物）、2-乙酰丙酮酸盐和乙酰丙酮（共价中间体类似物）和3）丙酮（产物）复合。这些结构将作为“快照”沿着酶的催化途径。将进行动力学实验以揭示限速步骤。这些将包括测量与合成底物和带有供电子和吸电子基团的酰化剂的反应速率。中间体捕获和溶剂交换实验将在突变体K116 R AAD酶（20%野生型活性）上进行，其中pKa扰动赖氨酸116已突变为精氨酸，以评估Schiff碱的限速步骤和/或稳定性是否发生变化。这些研究提供的信息将使我们能够评估酶的化学和结构贡献的稳定性和反应性的基本席夫碱中间体。这种方法最终可以扩展到不太明确的酶系统的分析。 这项研究的基础是对酶使用的基本方法的理解，这些方法可以将化学反应的速率提高10亿倍。拟议的研究将剖析一种酶，乙酰乙酸脱羧酶（AAD酶），以实现这样的速率加速所使用的化学步骤。将以两种方式检查化学步骤：1）通过比较AAD酶与其底物（通常与其相互作用的物质）和化学改变的底物的反应速度，以及2）通过测量由AAD酶促进的反应速度，其中参与化学反应的酶片段已使用分子生物学改变。然后，这些化学研究将与酶的三维结构的原子细节研究相关联。使用X射线晶体学方法，我们可以可视化组成蛋白质AAD酶的原子的确切位置，从而获得催化机器形状的详细图像。这些图片将被确定为AAD酶，单独和存在的物质称为抑制剂，类似于底物，但不能参与与酶的化学反应。此外，AAD酶与正常化学反应的终产物的相互作用可以通过该技术可视化。这些结构将作为“快照”沿着酶的途径。对AAD酶的结构和J~nct~on之间关系的理解最终可以用于理解其他酶促反应以及设计具有新功能的酶。 ***

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. ***