Mutation of Nicotinamide Pocket Residues in Rat Liver 3α-Hydroxysteroid Dehydrogenase Reveals Different Modes of Cofactor Binding†

Rat liver 3α-hydroxysteroid dehydrogenase (3α-HSD), an aldo−keto reductase, binds NADP+ in an extended anti-conformation across an (α/β)8-barrel. The orientation of the nicotinamide ring, which permits stereospecific transfer of the 4-pro-R hydride from NAD(P)H to substrate, is achieved by hydrogen bonds formed between the C3-carboxamide of the nicotinamide ring and Ser 166, Asn 167, and Gln 190 and by π-stacking between this ring and Tyr 216. These residues were mutated to yield S166A, N167A, Q190A, and Y216S. In these mutants, KdNADP(H) increased by 2−11-fold but without a significant change in KdNAD(H). Steady-state kinetic parameters showed that KmNADP+ increased 13−151-fold, and this was accompanied by comparable decreases in kcat/KmNADP+. By contrast, KmNAD+ increased 4−8-fold, but changes in kcat/KmNAD+ were more dramatic and ranged from 23- to 930-fold. Corresponding changes in binding energies indicated that each residue contributed equally to the binding of NADP(H) in the ground and transition s...