Mutagenesis of 3α-Hydroxysteroid Dehydrogenase Reveals a “Push−Pull” Mechanism for Proton Transfer in Aldo−Keto Reductases†

摘要: Rat liver 3α-hydroxysteroid dehydrogenase (3α-HSD, E.C. 1.1.1.213, AKR1C9) is a member of the aldo−keto reductase (AKR) superfamily which inactivates circulating steroid hormones. We have proposed catalytic mechanism in Tyr 55 acts as general acid with its pK value being lowered by hydrogen bond Lys 84 salt-linked to Asp 50. To test this mechanism, residues at active site were mutated and mutant enzymes (Y55F, Y55S, K84M, K84R, D50N, D50E, H117A) purified homogeneity from an Escherichia coli expression system. Spectrophotometric assays showed that mutations gave apparently inactive for oxidation reduction. All mutants appeared The efficiencies reduced 4−10-fold 50 300-fold H117A mutant. Fluorescence titration NADPH demonstrated each bound cofactor unimpeded. Equilibrium dialysis indicated ...