Analysis of the kinetic mechanism of haloalkane conjugation by mammalian theta-class glutathione transferases.

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 theta-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 C(2)H(5)Cl, CH(2)Br(2), or CH(2)Cl(2), 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 approximately 10-fold faster than C-Cl cleavage. The ratio k(cat)/K(m) is the most relevant parameter for issues of risk assessment.

Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays.

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity.

Molecular cloning and expression of rat liver bile acid CoA ligase.

Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3'-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates.