Identification of multiple target sites for a glutathione conjugate on glutathione-S-transferase by matrix-assisted laser desorption/ionization mass spectrometry.
A mass spectrometric method providing qualitative site-specific information regarding covalent modification of proteins is described. The method involves comparison of unmodified and modified proteins by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) peptide mapping in combination with site-specific mutagenesis of possible target amino acids. The approach is demonstrated through the mapping of glutathione-S-transferases (GSH transferases) before and after inhibition with the glutathione conjugate 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (GSTCBQ). The results demonstrate the utility of site-specific mutagenesis in combination with MALDI MS peptide mapping. Evidence is presented that three residues in or near the active site, including the hydroxyl groups of Tyr6 and Tyr115 and the sulphydryl group of Cys114, are target sites for GSTCBQ. Although only one GSTCBQ molecule per active site was detected, it appears to be distributed among all three target sites. In addition, MALDI MS peptide mapping covered 81% of the cDNA deduced amino acid sequence for GSH transferase and site-directed mutagenesis corresponding to a single amino acid substitution were verified.
Jespersen
S
S
Division of Analytical Chemistry, Center for Bio-Pharmaceutical Sciences, University of Leiden, The Netherlands.
Ploemen
J H
JH
van Bladeren
P J
PJ
Niessen
W M
WM
Tjaden
U R
UR
van der Greef
J
J
D001665:Binding Sites; D002703:Chloranil / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004791:Enzyme Inhibitors; D002703:Chloranil; C057105:2-gluthionyl-3,5,6-trichloro-1,4-benzoquinone; D005982:Glutathione Transferase; D005978:Glutathione