Homoserine transsuccinylase (HTS) catalyzes the conversion of homoserine to U- succinylhomoserine. This is the first specific step in the biosynthesis of methionine and S-adenosylmethionine by microorganisms. Due to the critical and specific functions in bacterial amino acid and co- factor biosynthesis, enzymes in this pathway present potential targets for broad spectrum antibacterial inhibitor design. Since the initial submission of this proposal, E. coli HTS has been cloned, over-expressed, and purified. The kinetic mechanism of HTS will be determined by a series of experiments. Experiments using variable concentrations of both substrates indicate that HTS employs a ping-pong mechanism. DTNP inhibits the enzyme, suggesting the presence of an active site thiol. We will investigate this possibility by labeling HTS with thiol modifying agents, such as vinyl pyridine, and identifying a labeled peptide. A detailed pH profile will be conducted to determine the ionization behavior of the group(s) that are involved in catalysis or substrate binding that are involved in catalysis or substrate binding. Initial protein crystallization attempts have identified conditions which produce microcrystals. We will optimize these conditions in an effort to grow diffraction quality crystals for three-dimensional structure determination. The crystals will be soaked with various substrates and inhibitors, which will define the active site and delineate important interactions. Time permitting, we will attempt to clone the functionally related protein, homoserine transacetylase, from either H. influenza or M. tuberculosis in order to conduct a comparative study between the two acyl transferases.
