Mapping the aspartic acid binding site of Escherichia coli asparagine synthetase B using substrate analogs.

Abstract

Novel inhibitors of asparagine synthetase, that will lower circulating levels of blood asparagine, have considerable potential in developing new protocols for the treatment of acute lymphoblastic leukemia. We now report the indirect characterization of the aspartate binding site of Escherichia coli asparagine synthetase B (AS-B) using a number of stereochemically, and conformationally, defined aspartic acid analogs. Two compounds, prepared using novel reaction conditions for the stereospecific beta-functionalization of aspartic acid diesters, have been found to be competitive inhibitors with respect to aspartate in kinetic studies on AS-B. Chemical modification experiments employing [(fluorosulfonyl)benzoyl]adenosine (FSBA), an ATP analog, demonstrate that both inhibitors bind to the aspartate binding site of AS-B. Our results reveal that large steric alterations in the substrate are not tolerated by the enzyme, consistent with the failure of previous efforts to develop AS inhibitors using random screening approaches, and that all of the ionizable groups are placed in close proximity in the bound conformation of aspartate.