Glutathione transferases (GSTs) are a family of enzymes that are key players in cellular detoxication. These enzymes catalyze the transfer of glutathione (GSH) to the electrophilic center of harmful compounds to promote their elimination.

The human Pi class (GST P1-1) is well-known for its overexpression in cancerous tissue and has been found to contribute to tumor growth and chemotherapeutic resistance. For these reasons, GST P1-1 has emerged as a promising therapeutic target to fight cancer by developing inhibitors and prodrugs (e.g. Telcyta) targeting the enzyme. GST P1-1 has also been suggested as a marker during carcinogenesis.

Apart from being cellular detoxicants some GSTs have come to develop other functions. One member of the human Alpha class, GST A3-3, plays an important role in steroid hormone biosynthesis by catalyzing the double-bond isomerization reaction of 5-androsten-3,17-dione and 5-pregnen-3,20-dione, precursors to the steroid hormones testosterone and progesterone. To date, in addition to the human enzyme, efficient ketosteroid isomerase activity has been identified in Alpha-class enzymes from equine and porcine tissues.

This thesis focuses on studying the Pi- and Alpha-class enzymes. In the first study, we characterize dog GST P1-1 and show that the enzyme shares certain class-specific similarities with the human enzyme in terms of substrate selectivity profile and inhibition profile. We also developed a thin-layer chromatography method to screen and semi-quantify Telcyta activity. In the second study, we show that the replacement of tyrosine¹⁰⁹ with histidine increased the activity with the anticancer prodrug Telcyta 2.9-fold, and we also show that the mutation Q85R positively influenced the thermostability of the enzyme. In the third study, we discovered a mutant enzyme, V2 (Q40M-E41Q-A46S-Y109H-V200L), with 22-fold higher catalytic efficiency than wildtype human GST P1-1 with cumene hydroperoxide. The mutation Y109H was responsible for a 10-fold increase in catalytic efficiency. In the fourth study, we discovered that GST A3-3 from the common marmoset monkey possessed prominent ketosteroid isomerase activity, albeit significantly lower than its human and equine counterparts, it was on par with porcine GST A2-2. In the fifth study, we solved the crystal structure of equine GST A3-3 in complex with the inhibitor triethyltin bromide. The structure reveals the interaction between triethyltin bromide, GSH, and Tyr⁹ in the enzyme.

All in all, the work presented in this thesis has added to the body of knowledge on the glutathione transferases from the Pi- and Alpha-classes.