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 labelling.
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-chloro2,4-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.
Hong
J L
JL
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Liu
L F
LF
Wang
L Y
LY
Tsai
S P
SP
Hsieh
C H
CH
Hsiao
C D
CD
Tam
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002703:Chloranil / Q000031:analogs & derivatives* / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D005982:Glutathione Transferase / Q000187:drug effects* / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010449:Peptide Mapping; D051381:Rats; D012694:Serine / Q000378:metabolism
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D002703:Chloranil; C057105:2-gluthionyl-3,5,6-trichloro-1,4-benzoquinone; D012694:Serine; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol