Functional mimicry of the active site of glutathione peroxidase by glutathione imprinted selenium-containing protein.
For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx.
Liu
Lei
L
State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130023, People's Republic of China.
Mao
Shi-zhong
SZ
Liu
Xiao-man
XM
Huang
Xin
X
Xu
Jia-yun
JY
Liu
Jun-qiu
JQ
Luo
Gui-min
GM
Shen
Jia-cong
JC
D001665:Binding Sites; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D005979:Glutathione Peroxidase / Q000378:metabolism*; D018716:Molecular Mimicry; D012643:Selenium / Q000378:metabolism*; D013379:Substrate Specificity
D005979:Glutathione Peroxidase; D012643:Selenium