A detailed examination of the structure/function relationships in the enzyme aspartate-beta-semialdehyde dehydrogenase (ASADH) is proposed. This enzyme plays a key role in the biosynthetic pathway leading to the amino acids lysine, isoleucine, methionine and threonine, and to essential components in bacterial cell wall biosynthesis.
The specific aims of this proposed research project are to (1) determine the detailed chemical mechanism of aspartate-beta-semialdehyde dehydrogenase, and to (2) determine the structure of the catalytic complexes of aspartate-beta-semialdehyde dehydrogenase. The primary tools that will be applied to this task are site-directed mutagenesis, kinetic and isotope effect studies to examine enzyme function, and x-ray crystallography and molecular modeling studies to determine the structure of significant enzyme-substrate, product, and intermediate complexes. The plan involves expanding the list of potential target amino acids for replacement by mutagenesis, kinetic and isotope effect studies to examine enzyme function, and x-ray crystallography. Molecular modeling studies will be employed to determine the structure of significant enzyme-substrate, product, and intermediate complexes. The asd gene sequence has been compared for homology with other enzymes that catalyze similar reactions, including the well characterized enzyme glyceraldehyde-3-phosphate dehydrogenase, and aspartate-beta-semi-aldehyde dehydrogenases from other species. Regions that have conserved amino acid sequences will be more likely to contain amino acid residues that are important for enzyme function, and have provided significant candidates for our mutagenesis studies. The recent determination of the three-dimensional structures of aspartate-beta-semialdehyde dehydrogenase by the Principal Investigator and his collaborators have provided constraints against which to judge and refine many of mechanistic hypotheses for this enzyme. This structural information has also suggested an expanded set of potential targets for mutagenesis studies to further investigate and refine the proposed mechanism, to identify substrate binding groups, to probe the conformation changes that are associated with enzyme catalysis, and to examine the details of subunit association.
| Viola, R E (2001) The central enzymes of the aspartate family of amino acid biosynthesis. Acc Chem Res 34:339-49 |
