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

被引:27
作者
Ma, HC [1 ]
Ratnam, K [1 ]
Penning, TM [1 ]
机构
[1] Univ Penn, Sch Med, Dept Pharmacol, Philadelphia, PA 19104 USA
关键词
D O I
10.1021/bi991659o
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 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 pi-stacking between this ring and Tyr 216. These residues were mutated to yield S166A, N167A, Q190A, and Y216S. In these mutants, K-d(NADP(H)) increased by 2-11-fold but without a significant change in K-d(NAD(H)). Steady-state kinetic parameters showed that K-m(NADP+) increased 13-151-fold, and this was accompanied by comparable 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 and transition states. However, the same residues stabilized the binding of NAD(H) only in the transition state. These observations suggest that different modes of binding exist for NADP(H) and NAD(H), Importantly, these modes were revealed by mutating residues in the nicotinamide pocket indicating that direct interactions with the 2'-phosphate in the adenine mononucleotide is not the sole determinant of cofactor preference. The single mutations were unable to invert or racemize the stereochemistry of hydride transfer even though the nicotinamide pocket can accommodate both anti- and syn-conformers once the necessary hydrogen bonds are eliminated. When 4-pro-R-[H-3]NADH 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. This observation coupled with the change in the binding energy for NAD(P)(H) in the transition state suggests that these mutants have altered the reaction trajectory for hydride transfer.
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页码:102 / 109
页数:8
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