Human Glutathione Transferase A3-3, a Highly Efficient Catalyst of Double-bond Isomerization in the Biosynthetic Pathway of Steroid Hormones

Abstract

The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Δ 5-androstene-3,17-dione and Δ5-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (kcat/Km) with Δ5-androstene-3,17-dione was determined as 5 × 10 6 M-1 s-1, which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 × 108 based on the ratio between kcat and the rate constant for the nonenzymatic isomerization of Δ 5-androstene-3,17-dione. Besides being high in absolute numbers, the kcat/Km value of GST A3-3 exceeds by a factor of ∼230 that of 3β-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Δ5-Δ4 double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones.