IDENTIFICATION OF AMINO-ACID-RESIDUES ESSENTIAL FOR HIGH AFLATOXIN B-1-8,9-EPOXIDE CONJUGATION ACTIVITY IN ALPHA-CLASS GLUTATHIONE S-TRANSFERASES THROUGH SITE-DIRECTED MUTAGENESIS

Mice constitutively express glutathione S-transferase mGSTA3-3 in live r. This isoform possesses uniquely high conjugating activity toward af latoxin B-1-8,9-epoxide (AFBO), thereby protecting mice from aflatoxin B-1-induced hepatocarcinogenicity. In contrast, rats constitutively e xpress a closely related GST isoenzyme, rGSTA3-3, with low AFBO activi ty and, therefore, are sensitive to aflatoxin B-1 exposure. Although t he two GSTs share 86% sequence identity and have similar catalytic act ivities toward 1-chloro-2,4-dinitrobenzene (CDNB), they have an approx imately 1000-fold difference in catalytic activity toward AFBO, To ide ntify amino acids that confer high activity toward AFBO, non-conserved rGSTA3-3 residues were replaced with mGSTA3-3 residues in two regions believed to form the substrate binding site. Twenty-one mutant rGSTA3 -3 enzymes were generated by site-directed mutagenesis using combinati ons of nine different residues, Except for the E208D mutant, single mu tations of rGSTA3-3 produced enzymes with no detectable AFBO activity. Generally, AFBO conjugation activity increased in additive fashion as mGSTA3-3 residues were introduced into the rGSTA3-3 enzyme with the s ix site mutant E104I/H108Y/Y111H/L207F/E208D/V217K displaying the high est AFBO activity (40 nmol/mg/min) of all the mutant enzymes, When thi s mutant enzyme was further modified by three additional substitutions (D103E/I105M/V106I) AFBO conjugation activity decreased 14-fold to 2. 8 nmol/mg/min. Although wild-type mGSTA3-3 AFBO conjugation activity ( 265 nmol/mg/min) could not be obtained by our rGSTA3-3 mutants, we wer e able to identify six mGSTA3-3 residues; Ile(104), Tyr(108), His(111) , Phe(207), Asp(208) and Lys(217) that, when collectively substituted into rGSTA3-3, substantially increased (>200-fold) glutathione conjuga tion activity toward AFBO, (C) 1998 Academic Press.