Dataset Information


Uncoupling intramolecular processing and substrate hydrolysis in the N-terminal nucleophile hydrolase hASRGL1 by circular permutation.

ABSTRACT: 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 (~50%), fettering the biophysical characterization 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 hASRGL1-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.


PROVIDER: S-EPMC3514461 | BioStudies | 2012-01-01

REPOSITORIES: biostudies

Similar Datasets

2016-01-01 | S-EPMC4809669 | BioStudies
2009-01-01 | S-EPMC2782781 | BioStudies
2012-01-01 | S-EPMC3289653 | BioStudies
2012-01-01 | S-EPMC3448794 | BioStudies
2013-01-01 | S-EPMC3633088 | BioStudies
2017-01-01 | S-EPMC5519988 | BioStudies
2009-01-01 | S-EPMC2755918 | BioStudies
2012-01-01 | S-EPMC3287067 | BioStudies
2007-01-01 | S-EPMC2631436 | BioStudies
2008-01-01 | S-EPMC2798589 | BioStudies