nitrophenols and cumene-hydroperoxide

We have previously expressed hexa-histidine-tagged human glutathione transferase GST T1-1 at very high levels in an Escherichia colilacZ mutagenicity assay strain. Ethylene dibromide (EDB), which is activated by GST T1-1, produces a potent response in the mutation assay. We have now constructed and expressed two SNP variants of wild-type GST T1-1:D141N and E173K. The EDB activation activities of both variant enzymes, as measured by the lacZ mutagenicity assay, are greatly reduced The D141N variant behaved similarly to the wild-type enzyme, in terms of expression level and specific activities for conjugation of glutathione with 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethylene diiodide (EDI), and 4-nitrobenzyl chloride (NBCl), and for peroxidative detoxication of cumene hydroperoxide (CuOOH). In contrast, variant E173K is poorly expressed, has no detectable activity with EPNP, NBCl, or CuOOH, and has EDI activity much lower than that of the wild-type enzyme. The circular dichroism (CD) thermal denaturation profiles of the wild-type protein and variant D141N show a sharp two-state transition between native and denatured states. Variant E173K showed a very different profile, consistent with improper or incomplete protein folding. Our results show that SNP variants can give rise to GSTT1-1 proteins with significantly altered properties.

Topics: Benzene Derivatives; Enzyme Stability; Epoxy Compounds; Genetic Variation; Glutathione Transferase; Humans; Hydrocarbons, Iodinated; Inactivation, Metabolic; Nitrobenzenes; Nitrophenols; Polymorphism, Single Nucleotide

Single nucleotide polymorphisms that cause amino acid substitutions in enzymes involved in the metabolism of xenobiotics can potentially have a significant effect on the efficacy and safety of therapeutic drugs.. We have utilized a bioinformatic approach to identify new polymorphisms in the glutathione transferase super family.. In this report we describe a P110S polymorphism in GSTA2 that occurs at a low frequency in Africans, Chinese and Europeans. The serine containing isoform has significantly diminished activity with a range of substrates including, delta-Androsten-3,17-dione, 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. The activity with cumene hydroperoxide may reflect a diminished physiological function since the glutathione peroxidase activity of GSTA2-2 plays a role in prostaglandin synthesis. In contrast, activity with p-nitrophenol acetate was significantly elevated. The position of this polymorphism in the active site and its effects on model substrates suggest that further investigation of its capacity to conjugate alkylating drugs is warranted.

Topics: Africa; Androstenedione; Benzene Derivatives; Binding Sites; China; Computational Biology; Diabetes Mellitus; Dinitrochlorobenzene; DNA; Europe; Glutathione Transferase; Humans; Kinetics; Models, Molecular; Nitrophenols; Polymorphism, Genetic; Recombinant Proteins