Site-directed mutagenesis studies of rat steroid 5α-reductase (isozyme-1): mutation of residues in the cofactor binding and C-terminal regions

Abstract

We have investigated the roles of highly conserved glycine (G175, G185), negatively charged (E188, D165) and histidine residues (H233, H237) in rat steroid 5α-reductase (isozyme-1), on NADPH, testosterone (T) binding and enzyme activity. The mutations G175R and G175S result in a two- to threefold increase in K m NADPH and an approximately fourfold decrease in the V max with no change in K m T. The mutation G185W resulted in a fivefold decrease in K m NADPH and an eightfold decrease in V max, with no change in K m T, whereas the mutations E188Q and D165N both resulted in inactive enzyme. Steady-state kinetic measurements showed that the mutation H233R resulted in an approximately 40-fold decrease in V max, an approximately 20-fold increase in K m T and no alteration in K m NADPH, whereas the mutation H237R resulted in virtually inactive enzyme. The results suggest that the conserved glycines are not essential for cofactor binding and activity, and that the negatively charged residues may contribute to enzyme stability, whereas the C-terminal histidines appear to be involved in substrate binding and catalytic activity.