U.S. Department of Energy

Pacific Northwest National Laboratory

Activity-Based Probes Developed to Understand Role of Glutathione S-transferases in Xenobiotic Detoxification

The cover of JACS.

Probes facilitate quantification of active enzyme-specific GST response following perturbation, indicative of the capacity for detoxification.

The Science                      

PNNL researchers developed and applied two new activity-based probes that irreversibly bind the active sites of glutathione S-transferases (GSTs) and report on their activity. GST enzymes are critical mediators of metabolism and detoxification in many mammalian organs, with highest levels of the enzymes found in the liver. The first probe was derived from the substrate glutathione, and it reports activity from the glutathione-binding portion of the GST site while the second probe targets the xenobiotic-binding site. By targeting both subsites with probes, researchers obtain a comprehensive look at GST activity and how it changes when exposed to perturbations such as high fat diets.

The Impact

Knowing how GST enzymes respond to physiological changes enables researchers to assess an individual’s susceptibility to xenobiotic (foreign chemical) exposures. GST activity-based probes can quantify the changes in function of their enzyme targets after exposure to chemicals, and they can quantify the enzyme-specific GST functional response to changes in physiology (e.g., obesity, disease).

Summary

Glutathione S-transferases (GSTs) are enzymes that catalyze the addition of glutathione to a wide variety of endogenous and exogenous substrates. These enzymes play highly important roles in the detoxification of various drug molecules as well as potent environmental carcinogens. Thus, the activity of these enzymes is critical for efficient metabolism and clearance of these toxicants. Until now, there haven’t been any methods to measure enzyme-specific GST activity. 

To address this need, PNNL researchers used an activity-based protein profiling (ABPP) approach that involves the use of small molecule probes to irreversibly bind, enrich, and facilitate the quantification of active enzymes of a specific function. The active site of GST enzymes consist of the glutathione-binding “g” site and the substrate binding “h” site. Since each site has a distinct function and individual contribution toward the overall activity of the enzyme, the individual activity of each site must be taken into account when investigating GST activity. To effectively and comprehensively quantify active GSTs, we synthesized and validated two activity-based probes that report on the activity of each subsite of the enzymes.  Using this approach, we also determined GST enzyme-specific impacts of obesity on intestinal GST enzymes.

Citation

E. Stoddard, B. Killinger, R. Nair, N. Sadler, R. Volk, S. Purvine, A. Shukla, J. Smith, and A. Wright, “Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-transferases.” J. Am. Chem. Soc. 139, 45 (2017). [DOI: 10.1021/jacs.7b07378].

Date: 
May 2018
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