Glutathione S-transferases comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo and xenobiotics. Although the total activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “H site”.  Therefore, we developed (1) a glutathione-based ABP (GSH-ABP) hypothesized to show activity for the “G site” and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. Quantitative proteomic analysis of GSH-ABP enriched enzyme targets finds labeling of several GST isoforms, the most active being GSTA4, GSTM1, and GSTM2. The GST-ABP is a derivative of a known mechanism-based inhibitor of GSTs that binds cysteine(s) within the GST active site and inhibits GST activity. Quantitative proteomic analysis of GST-ABP enriched targets reveals selective targeting of GSTT2. Validation of both probes and the corresponding confident GST targets was carried out by competition experiments. We show the ability of both probes to characterize GSTs in several mammalian tissues. We also show the utility of the probes in detecting changes to GST activity incurred due to obesity. <br/>

Functional characterization of glutathione S-transferases by photoreactive and mechanism-based activity-based probes

254th National Meeting (2017)

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