麩胱甘肽硫轉換酶 cGSTM1-1 與不同受質形成複合體之結構及功能研究

1. 麩胱甘肽硫轉換酶cGSTM1-1與不同受質形成複合體之結構及功能研究麩胱甘肽硫轉換酶cGSTM1-1與不同受質形成複合體之結構及功能研究 • 研究小雞肝臟的mu類麩胱甘肽硫轉換酶(glutathione S-transferase) cGSTM1-1，與麩胱甘肽(GSH)接合上1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP)形成的GS-EPNP作為受質形成複合體，進行X光結晶學研究以決定出結構，並修正解析度至2.8 A。其結構可解釋cGSTM1-1中epoxidase活性的因素。此複合體在結晶學上所定義之不對稱單位中含兩個dimer，依序定為AB及CD。在dimer AB中，EPNP 的部分朝向Arg107及Gln165且形成氫鍵。而在dimer CD中，EPNP的部分則伸出至由連接b1及a1的 loop和部分 C端殘基銜接形成的疏水性區，且 EPNP上的phenoxyl ring與Trp209側鏈形成強烈的環堆疊作用。我們因而推論出此兩種不同構形代表的EPNP分別接近其啟始及最終催化反應階段，此結論與cGSTM1-1突變株的酵素動力學實驗結果相互印證。

2. Structure and Functional Study of Glutathione S-transferase cGSTM1-1 Complexed with Various Substrates • We elucidated the three-dimensional structure of mu-class glutathione S-transferase cGSTM1-1 co-crystallized with the glutathionyl conjugated 1,2-epoxy-3-(p-nitrophenoxy)propane (GS-EPNP) at 2.8A resolution. The product found in the active site was 1-hydroxy-2-(S-glutathionyl)-3- (p-nitrophenoxy) propane, instead of the conventionally decided 2-hydroxy isomer. The structure can explain the epoxidase activity of cGSTM1-1 to the substrates. The EPNP moiety orients towards Arg107(α4 helix), and Gln165(α6 helix ) in dimer AB and protrudes into a hydrophobic region formed by the loop connectingβ1 and α1 and part of the C-terminal tail in dimer CD. The phenoxyl ring forms strong ring stacking with the Trp209 side-chain in dimer CD. We hypothesize that these two conformations represent the EPNP moiety close to the initial and final stages of the reaction mechanism, respectively. And the conclusion was confirmed with the report of the mutagenesis and kinetic studies of cGSTM1-1.