Uncoupling intramolecular processing and substrate hydrolysis in the N-terminal nucleophile hydrolase hASRGL1 by circular permutation.
Uncoupling intramolecular processing and substrate hydrolysis in the N-terminal nucleophile hydrolase hASRGL1 by circular permutation.
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DOI：
10.1021/cb300232n
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发表时间：
2012-11-16
影响因子：
4
通讯作者：
Zhang, Yan
中科院分区：
文献类型：
--
作者：
Li, Wenzong;Cantor, Jason R.;Yogesha, S. D.;Yang, Shirley;Chantranupong, Lynne;Liu, June Qingxia;Agnello, Giulia;Georgiou, George;Stone, Everett M.;Zhang, Yan
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.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
5.8
作者：
Dinndorf, Patricia Anne;Gootenberg, Joseph;Pazdur, Richard
通讯作者：
Pazdur, Richard
影响因子：
1.7
作者：
Michalska, Karohna;Jaskolski, Mariusz
通讯作者：
Jaskolski, Mariusz
影响因子：
4.8
作者：
Michalska, Karolina;Hernandez-Santoyo, Alejandra;Jaskolski, Mariusz
通讯作者：
Jaskolski, Mariusz
影响因子：
5.6
作者：
Michalska, Karolina;Bujacz, Grzegorz;Jaskolski, Mariusz
通讯作者：
Jaskolski, Mariusz