Catalytic Activity of Seleno-hGSTZ1c-1c Based on Site-directed Mutagenesis†

doi:10.7503/cjcu20140101

Abstract

Abstract:

Human glutathione transferase zeta 1c-1c(hGSTZ1c-1c) is considered to be an ideal protein scaffold for imitating glutathione peroxidase(GPx) owing to the natural binding site of glutathione(GSH). In this research, four cysteine(Cys) residues(Cys-137, Cys-154, Cys-165 and Cys-205) were mutated to serine(Ser) to avoid untargeted introduction of selenocysteine(Sec) residues, which could lead to structural change. Then Ser-14, Ser-15 and Ser-17 near the GSH binding site were mutated to Cys, respectively, and biosynthetically converted to Sec by a Cys auxotrophic expression system. Of those mutants, the seleno-containing mutants 15C, 14C/15C and 17C showed some GPx activities. Substitution of either Ser-14 or Ser-15 resulted in the loss of GST acitivity of hGSTZ1c-1c, indicating that Ser-14 and Ser-15 may play crucial roles in the reaction. And subsequent study suggested that Ser-14 may be essential in binding of GSH, while Ser-15 was probably involved in catalysis.

Key words: Site-directed mutagenesis, Glutathione peroxidase, Human glutathione transferase zeta

TrendMD:

GUO Xiao, YU Yang, CHEN Long, WANG Mingshuo, JIN Mengmeng, LIU Guangna, ZHANG Li, LI Tao, GAO Zhuang, WEI Jingyan. Catalytic Activity of Seleno-hGSTZ1c-1c Based on Site-directed Mutagenesis^†[J]. Chem. J. Chinese Universities, 2014, 35(10): 2109.

Figures/Tables 4

References 25

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Gene	Sequence 5'→3'^*
C137S-F	CCAGAACGCCATCACTTCTGGCTTTAACGCCCTGG
C137S-R	CCAGGGCGTTAAAGCCAGAAGTGATGGCGTTCTGG
C154S-F	GCACAGCGGGCATATACTCTGTAGGAGACGAGGTG
C154S-R	CACCTCGTCTCCTACAGAGTATATGCCCGCTGTGC
C165S-F	GACCATGGCTGATCTGAGCTTGGTGCCTCAGGTG
C165S-R	CACCTGAGGCACCAAGCTCAGATCAGCCATGGTC
C205S-F	CCAGGTGTCTCACCCCTCCCGGCAGCCAGATACAC
C205S-R	GTGTATCTGGCTGCCGGGAGGGGTGAGACACCTGG

Gene	Sequence 5'→3'^*
C137S-F	CCAGAACGCCATCACTTCTGGCTTTAACGCCCTGG
C137S-R	CCAGGGCGTTAAAGCCAGAAGTGATGGCGTTCTGG
C154S-F	GCACAGCGGGCATATACTCTGTAGGAGACGAGGTG
C154S-R	CACCTCGTCTCCTACAGAGTATATGCCCGCTGTGC
C165S-F	GACCATGGCTGATCTGAGCTTGGTGCCTCAGGTG
C165S-R	CACCTGAGGCACCAAGCTCAGATCAGCCATGGTC
C205S-F	CCAGGTGTCTCACCCCTCCCGGCAGCCAGATACAC
C205S-R	GTGTATCTGGCTGCCGGGAGGGGTGAGACACCTGG

Catalyst	GPx activity/(U·μmol^-1)	GST activity of protein/(μmol·min^-1·mg^-1)	Percentage of GST activity(%)
hGSTZ1c-1c ^[11]	ND	0.978±0.058	100
Seleno-hGSTZ1c ^[11]	ND	0.449±0.030	46.0
hGSTZ1c-1c^Ser	ND	0.575±0.026	58.8
Seleno-hGSTZ1c-1c^Ser	ND	0.489±0.017	50.0
hGSTZ1c-1c^Ser-14C	ND	0.016±0.008	1.6
Seleno-hGSTZ1c-1c^Ser-14C	ND	ND	ND
hGSTZ1c-1c^Ser-15C	ND	0.008±0.001	0.8
Seleno-hGSTZ1c-1c^Ser-15C	8.91±1.57	ND	ND
hGSTZ1c-1c^Ser-14C/15C	ND	0.018±0.006	1.8
Seleno-hGSTZ1c-1c^Ser-14C/15C	18.23±2.31	ND	ND
hGSTZ1c-1c^Ser-17C	ND	0.611±0.042	62.5
Seleno-hGSTZ1c-1c^Ser-17C	43.57±4.39	ND	ND

Catalyst	GPx activity/(U·μmol^-1)	GST activity of protein/(μmol·min^-1·mg^-1)	Percentage of GST activity(%)
hGSTZ1c-1c ^[11]	ND	0.978±0.058	100
Seleno-hGSTZ1c ^[11]	ND	0.449±0.030	46.0
hGSTZ1c-1c^Ser	ND	0.575±0.026	58.8
Seleno-hGSTZ1c-1c^Ser	ND	0.489±0.017	50.0
hGSTZ1c-1c^Ser-14C	ND	0.016±0.008	1.6
Seleno-hGSTZ1c-1c^Ser-14C	ND	ND	ND
hGSTZ1c-1c^Ser-15C	ND	0.008±0.001	0.8
Seleno-hGSTZ1c-1c^Ser-15C	8.91±1.57	ND	ND
hGSTZ1c-1c^Ser-14C/15C	ND	0.018±0.006	1.8
Seleno-hGSTZ1c-1c^Ser-14C/15C	18.23±2.31	ND	ND
hGSTZ1c-1c^Ser-17C	ND	0.611±0.042	62.5
Seleno-hGSTZ1c-1c^Ser-17C	43.57±4.39	ND	ND