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

ACS Chem Biol. 2012 Nov 16;7(11):1840-7. doi: 10.1021/cb300232n. Epub 2012 Aug 29.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidohydrolases / chemistry*
  • Amidohydrolases / genetics
  • Amidohydrolases / metabolism*
  • Asparaginase / chemistry*
  • Asparaginase / genetics
  • Asparaginase / metabolism*
  • Asparagine / metabolism
  • Autoantigens / chemistry*
  • Autoantigens / genetics
  • Autoantigens / metabolism*
  • Catalytic Domain
  • Crystallography, X-Ray
  • Enzyme Activation
  • Humans
  • Hydrolysis
  • Models, Molecular
  • Point Mutation
  • Protein Conformation

Substances

  • Autoantigens
  • Asparagine
  • Amidohydrolases
  • ASRGL1 protein, human
  • N-terminal nucleophile hydrolase
  • Asparaginase