CONJUGATION OF HIGHLY REACTIVE AFLATOXIN B-1 EXO-8,9-EPOXIDE CATALYZED BY RAT AND HUMAN GLUTATHIONE TRANSFERASES - ESTIMATION OF KINETIC-PARAMETERS

Aflatoxin B-1 (AFB(1)) exo-8,9-epoxide, the reactive product of the he patocarcinogen AFB(1), is stable in aprotic solvents but hydrolyzes ra pidly in H2O at 25 degrees C and pH 7 (t(1/2) = 1 s). However, it is a lso known that some glutathione (GSH) transferases can conjugate the e poxide with GSH to give the adduct in high yield. We developed an appr oach to estimating kinetic parameters for reactions involving this epo xide or other substrates that are unstable to H2O. Varying concentrati ons of the (anhydrous) epoxide and GSH transferase were mixed and the GSH conjugates were measured. The final concentrations of product were known for each set of the starting epoxide and enzyme concentrations in a modeling approach, where the competition with the hydrolysis reac tion is considered with two variables, a K for binding of the enzyme a nd epoxide and a rate k(2), which includes microscopic steps following complex formation and resulting in conjugate formation. The ratio k(2 )/K, a measure of enzyme efficiency, varied among individual recombina nt GSH transferases in the the order (rat) 10-10 >> 3-3 > (human) M1-1 > T1-1, A1-1 > P1-1 > A2-2, from 3 x 10(6) to 10 M(-1) s(-1). The hig h ratio of M1-1 among the human GSH transferase enzymes tested is cons istent with other work in which GSH-AFB(1) conjugates were not detecte d in hepatocytes with an M1 null polymorphism. This general kinetic ap proach should be applicable to estimation of kinetic parameters involv ed in the interaction of other unstable substrates with enzymes.