This project involves a continuation of our studies on the enzyme L-aspartase, which catalyzes the reversible addition of ammonia to fumaric acid. The long term objectives of this project are to acquire a detailed understanding, at the molecular level, of the events involved in the mechanism and the regulation of the reaction catalyzed by L-aspartase, and by several other aspartate-utilizing metalloenzymes. The urea cycle enzyme argininosuccinase catalyzes a related reaction involving the addition of arginine to fumaric acid. Partial loss of argininosuccinase activity has been implicated in an inborn error of metabolism in humans associated with mental deficiencies. The bifunctional enzyme aspartokinase- homoserine dehydrogenase also occupies a key position in metabolism, the commitment step to the biosynthesis of four amino acids starting from L-aspartic acid.
The specific aims proposed in this research plan are to: probe the detailed kinetic and chemical mechanism of this enzyme system; establish the role of metal ion activators and inhibitors; explore the dual role of L- aspartic acid as a substrate and an activator for the enzyme; and determine the structure and orientation of the binding sites on L- aspartase. The techniques that will be applied in order to address the specific aims of the research project will include equilibrium dialysis and kinetic binding studies of metal ions, substrate analogs, and activators. A range of kinetic studies, including initial velocity, product inhibition, pH, and isotope effect studies, will be utilized to elucidate dynamic information about the reaction catalyzed by L-aspartase. Magnetic resonance studies, including NMR paramagnetic relaxation studies to examine enzyme-- substrate and enzyme-activator interactions, NMR and EPR studies to examine metal ion interactions, low temperature NMR studies to characterize intermediates in the catalytic cycle, and x-ray crystallographic studies of enzyme-substrate and analog complexes, will be utilized to provide structural information about the interaction of metal ions, substrates and activators with L- aspartase. Taken together these techniques offer the opportunity to conduct a detailed examination of the events involved in the mechanism of L-aspartase and some related enzymes, and to examine the role played by metal ion and substrate effectors for these enzymes.
