Glutathione S-transferase 활성부위 잔기들의 역할에 관한 부위특이적 변이법

공광훈,조성희 中央大學校 遺傳工學硏究所 1995 遺傳工學硏究論集 Vol.8 No.1

GST의 활성중심 잔기는 Tyr7이다. Figure 3에 나타낸 바와 같이 Tyr7의 phenol성 수산기가 효소에 결합한 GSH의 thiol에 있는 proton의 해리를 촉진하여 친핵성을 높이는 것이 촉매기구의 가장 중요한 점이다. 또한 Arg13의 side chain들은 GSH의 thiolate anion에 대해 solvent로부터의 노출을 막아(탈수화) 반응성을 강화시킨다고 생각되어 진다. Lys44, Gln51, Gln64, Ser65, Asp98의 잔기들은 GSH의 결합에 중요하다. 특히 Gln64와 Asp98은 GSH의 결합에 필수적인 잔기로 여겨진다. In order to elucidate the roles of residues in the active site of glutathione S-trans-ferase(GST), seven residues in human GST P1-1 were individually replaced with phenylalanine, threonine or alanine by site-directed mutagenesis to obtain mutants Y7F, R13T, K44T, Q51A, Q64A, S65A and D98A. Enzymatic properties of mutants were compared with those of the wild-type enzyme. Tyr7 is considered to be important for catalytic activity in lowering the pKa of the thiol of GSH in the enzyme GSH complex. Arg13 seems to be essential for the enzymatic activity as mainly involved in the construction of a proper structure of the active site. Lys44, Gln51, Gln64, Ser65 and Asp98 were deduced to contribute to the binding of GSH.

인체 글루타티온 전달효소에 있는 알지닌 13 변이체의 기질 특이성에 관한 연구

안소연,공광훈 中央大學校 基礎科學硏究所 2001 基礎科學硏究所 論文集 Vol.15 No.-

In order to study the roole of residue in the active site of glutathione S-transferase(GST), Arg13 residue in human GST P 1-1 was replaced with alanine, leucine or lysine by site-directed mutagenesis to obtain mutants R13A, R13L and R13K. These mutants were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The specific activities were determined by measuring the initial rates of the enzymes-catalyzed conjugation of GSH towards electrophilic substrates. Our results suggest that Arg13 in human GST P1-1 contributes to the binding of electroophilic substrate, but it is not needed for the glutathione peroxidase activity and the steroid isomerase activity of human glutathione S-transferase p1-1.

Human tyrosinase 유전자의 대장균에서의 발현

박소영,공광훈,박희중 中央大學校 遺傳工學硏究所 1997 遺傳工學硏究論集 Vol.10 No.1

Tyrosinase는 활성부위에 한 쌍의 구리이온을 함유하고 있는 metalloprotein으로 멜라닌 생합성에 중요한 역할을 한다. Human tyrosinase의 메카니즘을 연구하기 위해 human tyrosinase 유전자에 대한 대장균에서의 대량발현계를 구축하였다. Plasmid pRHOHT2 (Shibahara et al, 1988. Tohoku J. Exp. Med. 156. 403)로부터 human tyrosinase cDNA sequence는 polymerase chain reaction에 의해 증폭되었으며, 발현벡터 pGEX-KG와 pTrcHis에 subcloning 되었다. 이렇게 작성된 plasmid pKG-Tyrosinase와 pHis-Tyrosinase를 대장균주 MV1190에 형질전환시켜, 유도제 isopropyl- β-D-thiogalactopyranoside로 human tyrosinase를 대량발현시켰다. 발현된 tyrosinase는 GSH affinity column과 immobilized metal affinity column으로 정제하였고, 정제된 재조합 tyrosinase는 SDS-gel 전기영동상에서 각각 84,000과 66,000 정도를 나타내었다. 또한 정제된 재조합 tyrosinase는 dopa oxidation 활성을 나타내었다. Tyrosinase is a bifunctional copper-containing metalloprotein and play a central role and of melanin biosynthesis. In the study, we constructed several E. coil-expression system of human tyrosinase using a cDNA encoding human tyrosinase for its mechanism study. Human tyrosinase cDNA sequence, plasmid pRHOHT2 (Shibahara et al. 1988, Tohoku J. Exp. Med. 156, 403), was amplified by the polymerase chain reaction and was subcloned with pGEX-KG and pTrcHis expression vector. These recombinant tyrosinases were produced by induction with isopropyl-β-D-thiogalactopyranoside. Overexpressed human tyrosinases were purified by GSH affinity column and immobilized metal affinity column. The molecular weights of the purified recombinant tyrosinases determined by SDS-polyacrylamide gel electrophoresis was about 84,000 (pKG-Tyrosinase) and 66,000 (pHis-Tyrosinase), respectively. These enzymes also showed dopa oxidation activity.

Human Glutathione S-transferase의 Tyr108 잔기의 역할에 관한 연구

박희중,공광훈 中央大學校 遺傳工學硏究所 1999 遺傳工學硏究論集 Vol.12 No.1

Glutathione S-transferase(GST, EC 2.5. 1.18)의 기질 결합부위에 대한 연구를 위하여 human GST P 1-1의 친전자성 기질 결합부위 (H-site)로 알려지고 있는 Tyr108 잔기에 대하여 부위특이적 변이법을 이용하여 Y108A, Y108F 그리고 Y108W의 새로운 변이체 3개를 얻었다. 대장균을 이용한 대량발현과 affinity column에 의해 정제된 변이체 효소에 대하여 GSH와 CDNB, DCNB, ETA, EPNP 또는 steroid와의 포합반응 활성을 측정하여 야생형과의 활성비교를 하였다. 야생형과의 활성비교에 있어서 각 변이체들의 변화를 볼 수 있었으며, 특히 입체적 장애를 준 Y108W 변이체의 경우 CDNB에 대한 활성이 다른 변이체에 비해 현저히 감소한 반면 ETA에 대한 활성은 증가하였다. 이러한 결과 Tyr108 잔기는 친전자성 기질 결합부위에 관련되는 잔기로 생각되어진다. In order to study the role of residue in the active site of glutathione S-transferase (GST), Tyr108 residue in human GST P 1-1 was replaced with alanine, phenylalanine and tryptophan by site-directed mutagenesis to obtain mutants Y108A, Y108F and Y108W. The specific activities were determined by measuring the initial rates of the enzymes-catalyzed conjugation of GSH with CDNB, DCNB, ETA, EPNP or steroid. The Y108W mutant slightly increased the conjugating activity toward ethacrynic acid(ETA)., but it decreased in specific activity toward 1-chloro-2,4-nitrobenzene(CDNB). On the other hand, the Y108A and Y108F mutants had negligible effect on the activity toward CDNB, ETA and DCNB of the wild type. These results indicated that Tyr108 in human GST P1-1 may contribute to the binding of electrophilic substrate.

Aspergillus niger 글루코오스 산화효소의 유도 발현

안소연,조현영,공광훈 中央大學校 基礎科學硏究所 2002 基礎科學硏究所 論文集 Vol.16 No.-

Glucose oxidase catalyzes the oxidation of β-D-glucose to gluconic acid. It is necessary to be obtained maximum glucose oxidase activity because the enzyme is used commercially for various applications, in particular extracelluar glucose oxidase. For high activity of extracellular glucose oxidase, we used the enzyme from Aspergillus niger and YEP medium. Extracellular glucose oxidase activity as high as 7.5Uml^-1 was obtained with sucrose as carbon source and peptone as nitrogen source. The result of testing a various of medium condition was obtainable the highest enzyme activity in condition of culture medium of 1% sucrose(w/v), 2% peptone(w/v) and 1% yeast extract(w/v) at 25℃. Our work indicate an economically attractive process for enzyme production. In addition Aspergillus niger glucose oxidase was purified by DEAE-Sephacel chromatography. The activity of the enzyme proceeded at pH 5.5 and the effective substrate of the enzyme was only glucose.

Thermomicrobium roseum 유래 Alcohol Dehydrogenase의 정제 및 특성

윤석영,홍민표,김은호,공광훈 중앙대학교 기초과학연구소 2000 基礎科學硏究所 論文集 Vol.14 No.-

Alcohol dehydrogenase from Thermomicrobium roseum was purified to electrophoretic homogeneity approximately 77-fold with a 38% activity yield by adenosine 5'-monophosphate and Mono Q column chromatography. The molecular weight of the enzyme was determined to be approximately 43,000 by SDS-polyacrylamide gel electrophoresis and 88,000 by gel chromatography, indicating a homodimeric structure. The Km and Vmax values of the enzyme for ethanol were 24.2 mM and 12.8 ㎛ol/min/mg, respectively. Pyrazole notably inhibited the enzymatic activity. Ca2+ ion activated the enzyme activity, but Cu2+ inhibited. The activity of the enzyme was optimal at pH 10.0 and 60℃.

Active-Site Mutants of Human Glutathione S-Transferase P1-1 : Effects of the Mutations on Substrate Specificity and Inhibition Characteristics

Kong,Kwang-Hoon,Park,Hee-Joong,Yoon,Suck-Young The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4

In order to gain further insight on the relationship between structure and function of glutathione S-transferase (GST), the six active-site mutants, R13T, K44T, Q51A, Q64A, S65A, and D98A, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The active-site mutants showed marked differences in substrate specificity. The substitution of Gln51 with theronine resulted in a drastic decrease in the specific activities to <10% of the wild-type value. The substitution of Arg13 with threonine resulted in more decreased specific activity toward cumene hydroperoxide and in the l50 values of S-(2,4-dinitrophenyl) glutathione and benanstatin A. These results suggest that the substitution of Arg13 with threonine changes the conformation of the active site to increase the affinity for the product or electrophilic substrate. Lys44 seems to be inthe vicinity of the H-site of hGST P1-1 or may contribute to some extents to the electrophile binding.

Poster Session 2 / G-29 : Site - Directed Mutagenesis of Amino Acid Residues Involved in the Electrophilic Substrate Binding of Human Glutathione S-Transferase P1-1

(Kwang Hoon Kong),(Hee Joong Park),(Hyang Jegal) 한국생화학분자생물학회 (구 한국생화학회) 1999 생화학분자생물학회 춘계학술발표논문집 Vol.- No.-

Poster Session 2 / G-9 : Expression and characterization of Bacillus Stearothermophilus Alcohol dehydrogenase in E. coli

(Kwang Hoon Kong),(Eun Jung Shim),(Eun Ho Kim) 한국생화학분자생물학회 (구 한국생화학회) 1999 생화학분자생물학회 춘계학술발표논문집 Vol.- No.-

Active - Site Mutants of Human Glutathione S-Transferase P1-1 : Effects of the Mutations on Substrate Specificity and Inhibition Characteristics

Kong, Kwang Hoon,Park, Hee Joong,Yoon, Suck Young 생화학분자생물학회 1999 BMB Reports Vol.31 No.4

In order to gain further insight on the relationship between structure and function of glutathione S-transferase (GST), the six active-site mutants, R13T, K44T, Q51A, Q64A, S65A, and D98A, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The active-site mutants showed marked differences in substrate specificity. The substitution of Gln51 with threonine resulted in a drastic decrease in the specific activities to $lt;10% of the wild-type value. The substitution of Arg13 with threonine resulted in more decreased specific activity toward cumene hydroperoxide and in the I_(50) values of S-(2,4-dinitrophenyl) glutathione and benanstatin A. These results suggest that the substitution of Arg13 with threonine changes the conformation of the active site to increase the affinity for the product or electrophilic substrate. Lys44 seems to be in the vicinity of the H-site of hGST P1-1 or may contribute to some extents to the electrophile binding.