The long term goal of this research project is to understand at a molecular level the chemical mechanism and regulation of the reaction catalyzed by L-aspartic acid ammonia lyase (aspartase). Many inborn metabolic disorders are the result of the low activity or the absence of a particular enzyme. In some of these diseases, a single point mutation is responsible for the dramatically altered enzyme reactivity. A knowledge of structure-function relationships at the molecular level is needed before deficiencies of this type can be understood, and before attempts can be made to modify these inactive enzymes using active site directed reagents or site directed mutagenesis. Aspartase has been chosen for detailed study for three reasons. First, it is a member of a large family of enzymes which use fumarate as a substrate. Relatively little mechanistic or structural information is known about the members of this family. Second, aspartase is a metalloenzyme which makes it well suited for structure function studies since the metal ion can act as a built-in probe of the structure. Finally, aspartase shows non-Michaelis-Menten kinetics above pH 7.5. The structural causes of such allosteric behavior are not well understood. A number or different techniques will be used to understand aspartase. Structural information will be derived from x-ray crystallography and from spectroscopic studies of native and mutant enzymes. Mechanistic information will come from both site directed mutagenesis combined with steady state kinetic studies and from chemical modification studies. Successful completion of this research proposal will yield a set of structures of all of the intermediates which occur during the chemical reaction and an improved understanding of the transformation between these intermediates.
