Structural and Functional Consequences of Haloenol Lactone Inactivation of Murine and Human Glutathione S-Transferase
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- 作者：Alyson E. Mitchell ; Jiang Zheng ; Bruce D. Hammock ; Mario Lo Bello ; and A. Daniel Jones
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：May 12, 1998
- 年：1998
- 卷：37
- 期：19
- 页码：6752 - 6759
- 全文大小：94K
- 年卷期：v.37,no.19(May 12, 1998)
- ISSN：1520-4995
文摘
Mass spectrometric analysis of proteolysis products ofhaloenol lactone-modified glutathioneS-transferase isozyme mGSTP1 indicates that the haloenollactone 3-cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone is covalently attached to the protein at Cys-47.Comparisons of the extent of adductformation with losses in enzymatic activity indicate that mGSTP1exhibits greatest reactivity toward thehaloenol lactone, followed by mGSTM1 and mGSTA3. Activities ofmGSTP1 and mGSTM1 decreasein inverse proportion to haloenol lactone concentration, whereasmodification had no apparent effect oncatalytic activity of mGSTA3. Decreases in activity agree with theextent of protein modification observedin ESI mass spectra for mGSTP1 and mGSTM1 but not for mGSTA3.Kinetic studies employingrecombinant human proteins with replacement of cysteine by serine atCys-47 and Cys-101 indicate thatrapid inactivation (t1/2 = 2 min) occursonly when residue 47 is cysteine. Mass spectra ofC47S-hGSTP1incubated with haloenol lactone demonstrate covalent attachment of ahaloenol lactone-glutathioneconjugate and suggest that an ester forms between the lactone andSer-47. Therefore, we propose thatinitial opening of the lactone ring is promoted by Cys-47 throughthioester formation between the lactonecarbonyl and the Cys-47 sulfhydryl. Enol-keto tautomerizationand enzyme-mediated hydrolytic cleavageof the thioester produces a reactive 
mages/gifchars/alpha.gif" BORDER=0>-bromoketone which reacts asecond time with Cys-47 and inactivatesthe enzyme. These results suggest that Pi class GSTs havethioesterase activity and that haloenol lactoneinactivation occurs through an enzyme-mediated process.