Catalytic Mechanism of Acetoacetate Decarboxylase

在羰基和胺亲核试剂之间形成席夫碱（亚胺）并失去水分是一种普遍的反应，在过去的五十年中，它作为酶催化反应的中间体的重要性已经变得很明显。本研究的长期目标是确定酶促进希夫碱中间体形成和反应活性的结构和化学机制。结合x射线晶体学，蛋白质化学和动力学的互补技术将被用作工具。提出的模型系统是乙酰乙酸脱羧酶（AADase）与其底物、产物和共价中间体或其稳定的类似物络合。该酶催化乙酰乙酸脱羧为丙酮和二氧化碳是一种“简单”脱羧酶，不需要辅助因子，而是利用酶Lysl 15的-氨基与pKa_6.0形成反应性共价加合物。将测定天然的、未络合的AADase酶的x射线晶体结构。几种酶配体配合物的结构也将得到解决：AADase将与1)2-氧丙烷磺酸盐（底物类似物）、2~乙酰丙酮酸酯和乙酰丙酮（共价中间类似物）和3)丙酮（产物）络合。这些结构将沿着酶的催化途径充当“快照”。动力学实验将揭示速率限制步骤(s)。这些将包括与合成底物和酰基化剂的反应速率的测量，这些试剂带有供电子和吸电子基团。将对突变体K1 16R AADase（20%野生型活性）进行中间捕获和溶剂交换实验，其中干扰Iysine 116的pKa突变为精氨酸，以评估是否在限速步骤和/或希夫碱稳定性方面发生了变化。这些研究提供的信息将使我们能够评估酶对基本希夫碱中间体的稳定性和反应性的化学和结构贡献。这种方法最终可以扩展到不太明确的酶系统的分析。这项拟议研究的基础是对酶用来将化学反应速率提高10亿倍的基本方法的理解。拟议的研究将剖析一种酶，乙酰乙酸脱羧酶（AADase）所使用的化学步骤，以实现这种速度加速。化学步骤将通过两种方式进行检查：1)通过比较AADase与其底物（通常与之相互作用的物质）的反应速度和化学改变的底物；2)通过测量AADase促进的反应速度，其中参与化学反应的酶片段已通过分子生物学被改变。然后，这些化学研究将与酶的原子细节三维结构的研究联系起来。利用x射线晶体学的方法，我们可以看到组成AADase蛋白的原子的确切位置，从而获得催化机制形状的详细图像。这样的图像将被确定为AADase，单独和存在称为抑制剂的物质，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可以通过该技术可视化。这些结构将沿着酶的路径充当“快照”。了解AADase的结构与J~nct~on之间的关系，最终可以用于理解其他酶的反应，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. ***