Catalytic Mechanism of Acetoacetate Decarboxylase

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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. ***