CHARACTERIZATION AND FUNCTIONAL-ANALYSIS OF THE CIS-AUTOPROTEOLYSIS ACTIVE-CENTER OF GLYCOSYLASPARAGINASE

Glycosylasparaginase is an N-terminal nucleophile hydrolase and is act ivated by intramolecular autoproteolytic processing. This cis-autoprot eolysis possesses unique kinetics characterized by a reversible N-O ac yl rearrangement step in the processing. Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved i n binding of free amino acids in the partially formed substrate pocket on certain mutant precursors. This binding site is sequestered in the wild-type precursor. Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the i ndividual mutants and amino acids. The polypeptide sequence at the pro cessing site, which is highly conserved, adopts a special conformation . Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket th rough interaction with Arg-180, The reactive nucleophile Thr-152 is ac tivated not only by deprotonation by His-150 but also by interaction w ith Thr-170, suggesting a His-Thr-Thr active triad for the autoproteol ysis.