title: Resonance energy transfer between sites in rat liver glutathione S-transferase, 1-1, selectively modified at cysteine-17 and cysteine-111.
abstract: Monobromobimane (mBBr) can label both Cys111 and Cys17 of rat liver glutathione S-transferase, 1-1 (GST 1-1). However, selective modification of Cys111 was achieved by the maleimide-based sulfhydryl reagents N-ethylmaleimide (NEM) and fluorescein 5-maleimide (NFM). Incubation of GST 1-1 with 5 mM NEM for 30 min at pH 7.5 and 25 degrees C leads to the formation of modified enzyme with 92% residual activity toward 1-chloro-2,4-dinitrobenzene and completely blocks Cys111 from subsequent reaction with either NFM or mBBr. Reaction of GST 1-1 with 0.2 mM NFM under the same conditions affords a modified enzyme with only 14% residual activity even though NFM and NEM target the same Cys111. The results indicate that when the bulky fluorescein is covalently bound to Cys111, the ligand projects into both the xenobiotic binding site and the glutathione site. After NEM or NFM modification of GST 1-1, the enzyme was further modified by monobromobimane at Cys17 with loss of activity. Together with the only tryptophan (Trp20), fluorescein linked to Cys111 and bimane to Cys17 provide three fluorescent probes to study the solution structure of GST 1-1. Fluorescence spectral analysis suggests that Trp20 and bimane linked to Cys17 are located in a relatively hydrophobic environment, while fluorescein linked to Cys111 is located in a charged environment. These fluorescent probes constitute three sets of donor-acceptor pairs for the measurement of fluorescence energy transfer, and distances calculated from such measurements are 20 A between Trp20 and bimane at Cys17, 19 A between Trp20 and fluorescein at Cys111, and < 22 A between bimane at Cys17 and fluorescein at Cys111. Molecular modeling studies indicate that fluorescein lies between the two subunits, is surrounded by charged residues, and is extended into the xenobiotic binding site. They also suggest that mBBr must approach from the dimer interface in order to reach the reaction site at Cys17.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
authors.forename: L
authors.initials: L
authors.lastname: Hu
authors.forename: R F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001643:Bridged Bicyclo Compounds; D003545:Cysteine / Q000737:chemistry*; D004735:Energy Transfer; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005982:Glutathione Transferase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents; D013820:Thermolysin
chemical_list: D001643:Bridged Bicyclo Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; C037041:fluorescein 5-maleimide; D005982:Glutathione Transferase; D013820:Thermolysin; D003545:Cysteine; D005033:Ethylmaleimide; C028381:monobromobimane