Uncoupling Intramolecular Processing and Substrate Hydrolysis in the N-Terminal Nucleophile Hydrolase hASRGL1 by Circular Permutation

The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of L-asparagine and isoaspartyl-dipeptides As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by. an intramolecular cleavage step with Thr168 as the catalytic residue However, in vitro, autoprocessing is incomplete (similar to 50%), fettering the biophysical characterization 1 of hASRGL1 We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled the autoprocessing reaction, allowing us to kinetically and structurally characterize this enzyme and the precursor like hASAGL1-Thr168Ala variant :Crystallographic and biochemical evidence suggest an activation mechanism where a torsional restraint on the Thr168 side chain helps drive the intramolecular processing reaction. Cleavage and formation of the active site releases the torsional restriction on Thr168, which is facilitated by a small conserved Gly-rich loop near the active site that allows the conformational changes necessary for activation.