RESONANCE ENERGY-TRANSFER BETWEEN SITES IN RAT-LIVER GLUTATHIONE-S-TRANSFERASE, 1-1, SELECTIVELY MODIFIED AT CYSTEINE-17 AND CYSTEINE-111

Manabromobimane (mBBr) can label both Cys(111) and Cys(17) of rat live r glutathione S-transferase, 1-1 (GST 1-1). However, selective modific ation of Cys(111) was achieved by the maleimide-based sulfhydryl reage nts N-ethylmaleimide (NEM) and fluorescein 5-maleimide (NFM). Incubati on of GST 1-1 with 5 mM NEM for 30 min at pH 7.5 and 25 degrees C lead s to the formation of modified enzyme with 92% residual activity towar d 1-chloro-2,4-dinitrobenzene and completely blocks Cys(111) from subs equent 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 1 4% residual activity even though NFM and NEM target the same Cys(111). The results indicate that when the bulky fluorescein is covalently bo und to Cys(111), 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 Cys(17) with loss of activity. Together with the only tryptophan (Trp(20)), fluores cein linked to Cys(111) and bimane to Cys(17) provide three fluorescen t probes to study the solution structure of GST 1-1. Fluorescence spec tral analysis suggests that Trp(20) and bimane linked to Cys(17) are l ocated in a relatively hydrophobic environment, while fluorescein link ed to Cys(111) is located in a charged environment. These fluorescent probes constitute three sets of donor-acceptor pairs for the measureme nt of fluorescence energy transfer, and distances calculated from such measurements are 20 Angstrom between Trp(20) and bimane at Cys(17), 1 9 Angstrom between Trp(20) and fluorescein at Cys(111), and <22 Angstr om between bimane at Cys(17) and fluorescein at Cys(111). Molecular mo deling studies indicate that fluorescein lies between the two subunits , is surrounded by charged residues, and is extended into the xenobiot ic binding site. They also suggest that mBBr must approach from the di mer interface in order to reach the reaction site at Cys(17).