Abstract
Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.
Highlights
GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1
The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction
The presence of GST A1-1 under conditions similar to those used for specific activity measurements, but in the absence of GSH, increased the rate of the isomerization about 16 times at 1 M enzyme concentration (Table I)
Summary
The GST-catalyzed isomerization of AD has not been subjected to detailed mechanistic studies despite its theoretical interest and its possible physiological significance. This GST reaction complements the activity of the bifunctional 3-hydroxysteroid dehydrogenase/⌬534isomerase (3-HSD/ Iso), but its relative efficiency and the role of GSH in catalysis have not previously been evaluated. In the present investigation the isomerization of AD catalyzed by the major human liver enzyme GST A1-1 has been studied in detail This abundant isoenzyme was shown to be significantly more active with AD than the isomerase component of the bifunctional 3-HSD/Iso previously described. Marked differences among related Alpha class GSTs in their ability to catalyze the isomerization of AD as well as the significance of an ionized thiol group of GSH acting as a base in the catalytic mechanism were demonstrated
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