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

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• 作者：Wenzong Li ; Jason R. Cantor ; S. D. Yogesha ; Shirley Yang ; Lynne Chantranupong ; June Qingxia Liu ; Giulia Agnello ; George Georgiou ; Everett M. Stone ; Yan Zhang
• 刊名：ACS Chemical Biology
• 出版年：2012
• 出版时间：November 16, 2012
• 年：2012
• 卷：7
• 期：11
• 页码：1840-1847
• 全文大小：388K
• 年卷期：v.7,no.11(November 16, 2012)
• ISSN：1554-8937

文摘

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.