Conjugation of highly reactive aflatoxin B1 exo-8,9-epoxide catalyzed by rat and human glutathione transferases: estimation of kinetic parameters.

Aflatoxin B1 (AFB1) exo-8,9-epoxide, the reactive product of the hepatocarcinogen AFB1, is stable in aprotic solvents but hydrolyzes rapidly in H2O at 25 degrees C and pH 7 (t1/2 = 1 s). However, it is also known that some glutathione (GSH) transferases can conjugate the epoxide with GSH to give the adduct in high yield. We developed an approach to estimating kinetic parameters for reactions involving this epoxide or other substrates that are unstable to H2O. Varying concentrations 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 reaction is considered with two variables, a K for binding of the enzyme and epoxide and a rate k2, which includes microscopic steps following complex formation and resulting in conjugate formation. The ratio k2/K, a measure of enzyme efficiency, varied among individual recombinant 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 high ratio of M1-1 among the human GSH transferase enzymes tested is consistent with other work in which GSH-AFB1 conjugates were not detected in hepatocytes with an M1 null polymorphism. This general kinetic approach should be applicable to estimation of kinetic parameters involved in the interaction of other unstable substrates with enzymes.