CHEMICAL MODIFICATION OF GLUTATHIONE-S-TRANSFERASE FROM C6 36, AN AEDES-ALBOPICTUS CELL-LINE/

The cytosolic glutathione S-transferase from an Aedes albopictus cell line, C6/36, was sensitive to tetranitromethane, phenylglyoxal or pyri doxal phosphate modification, but was unaffected by N-acetylimidazole or diethyl pyrocarbonate. The extent of inactivation of the enzyme by those sensitive reagents was dependent on reagent concentrations but w as biphasic in nature and did not follow pseudo-first-order kinetics, Glutathione, ethacrynic acid, S-(hexyl)glutathione, or S-(2,4-dinitrop henyl)glutathione gave substantial protection of the enzyme from inact ivation by these reagents. The modified enzyme showed varying Michaeli s constants for glutathione (K-mGSH) or 1-chloro-2,4-dinitrobenzene (K -mCDNB) and a smaller catalytic constant (k(cat)). These results indic ate the involvement of tyrosine, arginine and lysine residues in the r eaction mechanism of the mosquito C6/36 cell glutathione S-transferase . From the results of chemical modification and previous pH studies [C hang G.-G., Tsai L.-N., Tang S.-S., and Wang T.-C. (1994) Arch. Bioche m. Biophys. 310, 134-143], we propose that tyrosine residue functions as a general base, promoting ionization of the thiol group of enzyme-b ound glutathione and resulting in formation of a more reactive nucleop hile thiolate that facilitates conjugation. The essential arginine and lysine residues may participate in maintenance of the correct active center structure.