Escherichia coli asparaginase II catalyzes the hydrolysis of l-asparagine to l-aspartate via a threonine-bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threomines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 Å resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.
