Analysis of the Kinetic Mechanism of Haloalkane Conjugation by Mammalian θ-Class Glutathione Transferases

Abstract

Glutathione (GSH) transferases (GSTs) catalyze the conjugation of small haloalkanes with GSH. In the case of dihalomethanes and vic-1,2-dihaloalkanes, the reaction leads to the formation of genotoxic GSH conjugates. A generally established feature of the reaction of the mammalian θ-class GSTs, which preferentially catalyze these reactions, is the lack of saturability of the rate with regard to the substrate concentration. However, the bacterial GST DM11 catalyzes the same reactions with a relatively low K_m. Recently, DM11 has been shown to exhibit burst kinetics, with a rate-determining k_off rate for product (Stourman et al. (2003) Biochemistry 42, 11048−11056). We examined rat GST 5-5 and human GST T1-1 and did not detect any burst kinetics in the conjugation of C₂H₅Cl, CH₂Br₂, or CH₂Cl₂, distinguishing these enzymes from GST DM11. The kinetic results were fit to a minimal mechanism in which the rate-limiting step is halide displacement. The differences in the steady state kinetics of conjugations catalyzed by bacterial GST DM11 and the mammalian GSTs 5-5 and T1-1 are concluded to be the result of differences in the rate-limiting steps and not to inherent enzyme affinity for the haloalkanes. The results may be interpreted in the context of a model in which the halide order affects the rate of carbon−halogen bond cleavage of all such reactions catalyzed by the GSTs. With GST DM11, the halide order is manifested in the K_m parameter but not k_cat. With mammalian GSTs, the high K_m is difficult to estimate. With all of the GSTs, the halide order is seen in the enzyme efficiency, k_cat/K_m, with C−Br cleavage ∼10-fold faster than C−Cl cleavage. The ratio k_cat/K_m is the most relevant parameter for issues of risk assessment.