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공광훈,Jong-Uk Koh,So-Youn Ahn,Hee-Joong Park 대한화학회 2005 Bulletin of the Korean Chemical Society Vol.26 No.3
To gain further insight on the relationship between structure and functions of glutathione S-transferase (GST), the three tyrosine 108 mutants, Y108A, Y108F, and Y108W, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitution of Tyr 108 with alanine resulted in significant decrease of the GSH-conjugation activity and the GSH peroxidase activity, but approximately 63% increase of steroid isomerase activity toward 5–androstene 3,17-dione. On the other hand, the substitution of Tyr 108 with phenylalanine resulted in decreases of kcat and kcat /KmEPNP by 2 orders of magnitude, suggesting that Tyr 108 residue of hGSTP1-1 are considered to be important for the catalysis and the binding of the epoxide substrates. The substitution of Tyr 108 with tryptophan resulted in significant decreases of the specific activities toward EPNP, cumene hydroperoxide and 5–androstene 3,17-dione, but approximately 2-fold increase on the enzyme-catalyzed addition of GSH to DCNB. We conclude from these results that Tyr 108 in hGST P1-1 plays very different roles depending upon the nature of the electrophilic substrates.
공광훈,박희중,이희진 한국분석과학회 2009 분석과학 Vol.22 No.3
To gain further insight into herbicide detoxification of plant, we purified a glutathione S-transferase from Brassica oleracea (BoGST) and studied its substrate specificity towards several xenobiotic compounds. The BoGST was purified to electrophoretic homogeneity with approximately 10% activity yield by DEAE-Sephacel and GSHSepharose column chromatography. The molecular weight of the BoGST was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure. The activity of the BoGST was significantly inhibited by S-hexyl-GSH and S-(2,4-dinitrophenyl)GSH. The substrate specificity of the BoGST displayed high activities towards CDNB, a general GST substrate and ethacrynic acid. It also exhibited GSH peroxidase activity toward cumene hydroperoxide.
Bacillus stearothermophilus alcohol dehydrogenase 유전자의 대장균에서의 발현
심은정,김용태,공광훈 中央大學校 遺傳工學硏究所 1998 遺傳工學硏究論集 Vol.11 No.1
Bacillus stearothermopilus NCA1503로부터 PCR을 이용하여 ADH 유전자를 증폭시킨 후 발현 벡터인 pGEX-KG에 삽입시켜 glutathion S-transferase와 융합 단백질로 대장균에서 대량 발현시켰다. 재조합 GST-ADH는 37℃에서 1mM의 IPTG로 단백질의 발현을 유도시켰으며, 발현된 단백질을 GSH-affinity컬럼을 이용하여 한번에 정제하였다. 정제된 GST-ADH의 특성조사를 통하여 발현된 융합 단백질은 본래의 효소와 유사한 생화학적 특성을 나타내었으며, 보다 안정하다는 것을 알 수 있었다. Bacillus stearothermopilus NCA1503 is an ethanologenic facultative anaerobic thermophile with an optumal growth temperature of 55℃. These strain was found to produce an thermostable alcohol dehydrogenase (ADH-T) amounting to 1 to 2% of soluble cell protein. The ADH-T gene from Bacillus stearothormopilus was amplified by the polymerase chain reaction. The amplified DNA was inserted into the expression vector pGEX-KG, and expressed it as a fusion protein with glutathione-S-transforase in E coli. The recombinant GST-ADH was produced by induction with 1mM IPTG at 37℃ and purified by GSH affinity column in a single step. The affinity-purified GST-ADH shows biochemical properties analogous to those of the native enzyme and is highly stable against high temperature.
Glutathione S-transferase의 기질결합부위에 관한 연구
박희중,김용태,공광훈 中央大學校 遺傳工學硏究所 1998 遺傳工學硏究論集 Vol.11 No.1
Glutathione S-transferase(GST, EC 2.5. 1.18)의 기질 결합부위에 대한 연구를 위하여 human GST P 1-1의 6개의 잔기에 대하여 부위특이적 변이법을 이용하여 F8A, V10A, N204A, G205A, G205I 그리고 G205V의 새로운 변이체 6개를 얻었다. 대장균을 이용한 대량발현과 affinity column에 의해 정제된 변이체 효소에 대하여 GSH와 CDNB, DCNB 또는 ETA와의 포합반응 활성을 측정하였다. 야생형과의 활성비교에 있어서 G205V 변이체의 경우 CDNB나 ETA에 대한 활성이 현저히 감소함을 알 수 있었다. 이러한 결과 Gly205 잔기는 기질결합부위의 중요한 잔기로 생각되어진다. In order to study on the substrate binding site of glutathione S-transferase(GST), six residues in human GST P 1-1 were individually replaced with alanine, isoleucine or valine by site-directed mutagenesis to obtain mutants F8A, V10A, N204A, G205A, G205I and G205V. The specific activities were determined by measuring the initial rates of the enzymes-catalyzed conjugation of GSH with CDNB, DCNB or ETA. The replacement of Gly205 with valine resulted in the drastic decreases in the specific activities toward CDNB and ETA. These results suggest that Gly205 is an important residue in the binding site of the xenobiotic substrate.
Rice glutathione S-transferase의 정제 및 특성
박희중,공광훈 中央大學校 遺傳工學硏究所 1996 遺傳工學硏究論集 Vol.9 No.1
Rice로부터 DEAE-Sepharose와 GSH- garose 및 Sephadex G-75 column chromatography를 이용하여 GST를 분리정제하였다. 정제된 효소는 분자량이 전기동영상에서는 약 28 kDa, gel chromatography상에서는 약 54kDa인 homodimeric 구조이다. 또한 정제된 효소는 다른 종 GST들에 공통적인 특징인 1-chloro-2,4-dinitrobenzene(CDNB)에 대해 높은 기질특이성을 보였으며, S-hexylglutathione과 benanstatin A 에 대해서도 저해효과를 보였다. 효소의 최적온도는 45℃ 를 나타내었다. Glutathione S-transferase (GST) was purified from rice by DEAE-Sepharose, GSH-agarose and Sephadex G-75 column chromatography. The molecular mass of the purified GST was determined to be 54,000 by gel chromatography and 28,000 by SDS-polyacrylamide gel electrophoresis, indicating a homodimeric structure. The enzyme had high substrate activity of the enzyme toward the substrate CDNB was inhibited by S-hexylglutathione and benanstatin A. The optimun pH of enzyme was 7.0 and the optimum temperature was 45℃ with CDNB as a substrate.