[논문]Functional Studies of Tyrosine 108 Residue in the Active Site of Human Glutathione S-Transferase P1-1

Park, Hee-Joong; Koh, Jong-Uk; Ahn, So-Youn; Kong, Kwang-Hoon

doi:10.5012/bkcs.2005.26.3.433

Functional Studies of Tyrosine 108 Residue in the Active Site of Human Glutathione S-Transferase P1-1 원문보기

Bulletin of the Korean chemical society, v.26 no.3, 2005년, pp.433 - 439

Park, Hee-Joong (Department of Chemistry, College of Sciences, Chung-Ang University) , Koh, Jong-Uk (Department of Chemistry, College of Sciences, Chung-Ang University) , Ahn, So-Youn (Department of Chemistry, College of Sciences, Chung-Ang University) , Kong, Kwang-Hoon (Department of Chemistry, College of Sciences, Chung-Ang University)

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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 ${\Delta}^5$–[androstene 3,17-dione. On the other hand, the substitution of Tyr 108 with phenylalanine resulted in decreases of $k_{cat}\;and\;k_{cat}/K_m{^{EPNP}}$ 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 ${\Delta}^5$–ndrostene 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.

주제어

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대상 데이터

S-(2,4-Dinitrophenyl)glutathione was synthesized by the method of Schramm et al.³⁴ Glutathione Sepharose was purchased from Pharmacia Biotech (Uppsala, Sweden). All other reagents used were of the highest grade commercially available.

성능/효과

The substitution of Tyr 108 with alanine significantly affected GSH-conjugation activity, GSH peroxidase activity and steroid isomerase activity. The substitution of Tyr 108 with alanine resulted in approximately 40-60% decrease of the specific activities toward DCNB and EPNP and 98% decrease of the GSH peroxidase activity toward cumene hydroperoxide (Table 1 and 2). This substitution also resulted in large increases of K_m values toward electrophilic substrates (Table 5 and 6) and a significant decrease of I₅₀ value for hematin (Fig.
The substitution of Tyr 108 with alanine resulted in approximately 40-60% decrease of the specific activities toward DCNB and EPNP. The substitution of Tyr 108 with phenylalanine had negligible effect on the specific activity toward DCNB, but it resulted in approximately 90% decrease of the specific activity toward EPNP. The substitution of Tyr 108 with tryptophan resulted in approximately 60% decrease of the specific activities toward EPNP, but it resulted in approximately 200% increase of the specific activity toward DCNB.
Table 7 summarizes the kinetic parameters of the mutant enzymes for steroid isomerase activity. The substitution of Tyr 108 with tryptophan resulted in a 2-fold decrease of K_m^GSH, whereas K_m^STEROID value resulted in a 7.6-fold increase. On the other hand, the K_m^GSH values of the Y108A and Y108F were similar to that of the wild type.
The substitution of Tyr 108 with phenylalanine had negligible effect on the specific activity toward DCNB, but it resulted in approximately 90% decrease of the specific activity toward EPNP. The substitution of Tyr 108 with tryptophan resulted in approximately 60% decrease of the specific activities toward EPNP, but it resulted in approximately 200% increase of the specific activity toward DCNB.

후속연구

The results show that Tyr 108 plays very different roles depending upon the nature of the electrophilic cosubstrate. This study offers the information on the precise enzyme-substrate interactions responsible for the catalytic properties of hGST P1-1, and it will be of great value in designing new inhibitors that may prove useful in chemotherapy and new enzymes having different substrate specificity.

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