MODIFICATION OF GLUTATHIONE-S-TRANSFERASE-3-3 MUTANTS WITH 2-(S-GLUTATHIONYL)-3,5,6-TRICHLORO-1,4-BENZOQUINONE - IDENTIFICATION OF THE C-TERMINAL TRYPTIC FRAGMENT AS PART OF THE H-SITE AND EVIDENCE THAT 2-(S-GLUTATHIONYL)-3,5,6-TRICHLORO-1,4-BENZOQUINONE IS NOT SPECIFIC FOR CYSTEINE LABELING

被引:9
作者
HONG, JL [1 ]
LIU, LF [1 ]
WANG, LY [1 ]
TSAI, SP [1 ]
HSIEH, CH [1 ]
HSIAO, CD [1 ]
TAM, MF [1 ]
机构
[1] ACAD SINICA, INST MOL BIOL, TAIPEI 11529, TAIWAN
关键词
D O I
10.1042/bj3040825
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A triple mutant of rat liver glutathione S-transferase 3-3 that has all three cysteine residues replaced with serine (CallS) and a quadruple mutant with a Tyr-115 to phenylalanine substitution on CallS (CallSY115F) were reacted with 2-(S-glutathionyl)-3,5,6-trichloro-1 ,4-benzoquinone (GS-1,4-TCBQ). The modified proteins were analysed on a triple-quadrupole mass spectrometer equipped with an electrospray ionization source. At an enzyme: GS-1,4-TCBQ ratio of 1:10, the enzymes were modified at multiple sites. Covalent attachment of a single inhibitor on to the protein was achieved by lowering the enzyme:GS-1,4-TCBQ ratio to 1:1. Results from m.s. analyses suggest that the inhibitor on the CallSY115F mutant exists as a glutathionyl dichlorobenzoquinone derivative. The modifiers of the CallS mutants are glutathionyl monochlorobenzoquinone derivatives. Therefore, GS-1,4-TCBQ reacts at a single site on CallSY115F, but probably cross-links two regions on wild-type and CallS mutant. To confirm our observation, CallS was modified with 1-chloro-2,4-dinitrobenzene, which. specifically labels Tyr-115, before reacting with GS-1,4-TCBQ. The inhibitor formed a glutathionyl dichlorobenzoquinone adduct on the dinitrophenyl-CallS mutant. In addition, the benzoquinone derivative on the protein can be partially removed by 1-chloro-2,4-dinitrobenzene. Peptide mapping and sequencing analysis of the GS-1,4-TCBQ-modified CallS mutant revealed that the C-terminal 16-amino-acid fragment is labelled. Molecular modelling suggests the C(5) and C(6) on the benzoquinone ring of the inhibitor interact with the oxygen atoms of Tyr-115 and Ser-209 respectively.-
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页码:825 / 831
页数:7
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