Mutation of nicotinamide pocket residues in rat liver 3 alpha-hydroxysteroid dehydrogenase reveals different modes of cofactor binding

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), an aldo-keto reductase, binds NADP(+) in an extended anti-conformation across an (alpha/ beta)(8)-barrel. The orientation of the nicotinamide ring, which permits st ereospecific 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 nicotin amide ring and Ser 166, Asn 167, and Gln 190 and by pi-stacking between thi s ring and Tyr 216. These residues were mutated to yield S166A, N167A, Q190 A, and Y216S. In these mutants, K-d(NADP(H)) increased by 2-11-fold but wit hout a significant change in K-d(NAD(H)). Steady-state kinetic parameters s howed that K-m(NADP+) increased 13-151-fold, and this was accompanied by co mparable decreases in k(cat)/K-m(NADP+). By contrast, K-m(NAD+) increased 4 -8-fold, but changes in k(cat)/K-m(NAD+) 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 an d transition states. However, the same residues stabilized the binding of N AD(H) only in the transition state. These observations suggest that differe nt modes of binding exist for NADP(H) and NAD(H), Importantly, these modes were revealed by mutating residues in the nicotinamide pocket indicating th at direct interactions with the 2'-phosphate in the adenine mononucleotide is not the sole determinant of cofactor preference. The single mutations we re unable to invert or racemize the stereochemistry of hydride transfer eve n though the nicotinamide pocket can accommodate both anti- and syn-conform ers once the necessary hydrogen bonds are eliminated. When 4-pro-R-[H-3]NAD H was used to monitor incorporation into [C-14]-5 alpha-dihydrotestosterone , a decrease in the H-3:C-14 ratio was observed in the mutants relative to wild-type enzyme reflecting a pronounced primary kinetic isotope effect. Th is observation coupled with the change in the binding energy for NAD(P)(H) in the transition state suggests that these mutants have altered the reacti on trajectory for hydride transfer.