The goal of this work is to understand the allosteric effects at the molecular level on the activity of the enzyme, L-aspartase, which catalyzes the reversible deamination of L-aspartic acid to form fumarate. L-aspartase is a member of a family of enzymes that have fumarate as a substrate. The enzyme contains an allosteric activator site, which binds the substrate, L-aspartic acid, as well as a series of analogs. The enzyme has the interesting property that the presence of an activator is only required at basic pH. At low pH the activator site appears decoupled from catalytic operation. The location and structure of the activator site is unknown and the mechanism by which an activator affects the enzyme activity is not understood.
The specific aims of the work are: to identify the activator binding site by determining the amino acids that interact with the activator, determine the orientation of the activator in the binding site, and measure the distance between the activator and the active sites. The primary technique to be used is magic-angle-spinning solid-state NMR. Inter-nuclear distances will be measured using recently developed dipolar recoupling techniques of REDOR, DRAMA, and SEDRA. Distances will be measured between at the activator binding site between the enzyme amino acids and the carbon, nitrogen, or phosphorous of several activators. In addition to measuring distance, the NMR results will also detail the type of amino acids that form the activator binding site. By using this information, along with the activator-protein distances, a model for the binding site will be constructed. By measuring the distances between a bound substrate analog and a bound activator, the inter-site distance will be determined. With knowledge of the location and structure of the activator binding site the structural changes that occur in the enzyme upon activator binding will be investigated and the role of the activator understood.
