Functional analysis of Tyr7 residue in human glutathione S-transferase P1-1

Abstract

In order to clarify the functional role of Tyr7 in human glutathione S-transferase P1-1, we extensively investigated the effect of mutation of Tyr7 on the substrate specificity and inhibition characteristics. The mutational replacement of Tyr7 with phenylalanine lowered the specific activities with 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy) propane for GSH-conjugation reaction to 3~5% of the values for the wild-type enzyme. The pKa of the thiol group of GSH bound in Y7F was about 2.4 pK units higher than that in the wild-type enzyme. The I(50) of hematin for Y7F was similar to that for the wild-type enzyme and those of benastatin A and S-(2,4-dinitrophenyl)glutathione were only moderately decreased. These results suggest that Tyr7 is considered to be important the catalytic activities not only for GSH-chloronitrobenzene derivatives but also for GSH-epoxide conjugation reaction, rather than to binding of the substrates.

본 실험은 human glutathione S-transferase P1-1의 tyrosine 7 잔기에 대한 변이체를 작성하고, 기질특이성과 저해제의 효과를 조사하여, 이 잔기의 기능을 분석한 것이다. 1,2-dichloro-4-nitrobenzene과 1,2-epoxy-3-(p-nitrophenoxy)propane에 대한 GSH 포합반응에 대한 활성은 야생형에 비해 변이체 Y7F에서는 3~5%로 크게 저하하였으며, 효소에 결합한 GSH의 thiol기의 pKa는 2.4 pK 높았다. 저해제 hematin에 대한 I(50)값은 야생형과 변이체 Y7F에서 비슷하게 나타났으며, 저해제 benastatin A와 S-(2,4-dinitrophenyl) glutathione에 대한 I(50)값들은 다소 감소하였다. 이러한 결과들로부터 tyrosine 7 잔기는 기질의 결합에 관여하기보다 GSH-chloronitrobenzene 유도체와 GSH-epoxide 포함반응에 대한 촉매활성에 중요하다고 생각된다.