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• W2060603106 abstract "Aldo-keto reductase (AKR1C) isoforms can regulate ligand access to nuclear receptors by acting as hydroxysteroid dehydrogenases. The principles that govern steroid hormone binding and steroid turnover by these enzymes were analyzed using rat 3α-hydroxysteroid dehydrogenase (3α-HSD, AKR1C9) as the protein model. Systematic alanine scanning mutagenesis was performed on the substrate-binding pocket as defined by the crystal structure of the 3α-HSD·NADP+·testosterone ternary complex. T24, L54, F118, F129, T226, W227, N306, and Y310 were individually mutated to alanine, while catalytic residues Y55 and H117 were unaltered. The effects of these mutations on the ordered bi-bi mechanism were examined. No mutations changed the affinity for NADPH by more than 2−3-fold. Fluorescence titrations of the energy transfer band of the E·NADPH complex with competitive inhibitors testosterone and progesterone showed that the largest effect was a 23-fold decrease in the affinity for progesterone in the W227A mutant. By contrast, changes in the Kd for testosterone were negligible. Examination of the kcat/Km data for these mutants indicated that, irrespective of steroid substrate, the bimolecular rate constant was more adversely affected when alanine replaced an aromatic hydrophobic residue. By far, the greatest effects were on kcat (decreases of more than 2 log units), suggesting that the rate-determining step was either altered or slowed significantly. Single- and multiple-turnover experiments for androsterone oxidation showed that while the wild-type enzyme demonstrated a klim and burst kinetics consistent with slow product release, the W227A and F118A mutants eliminated this kinetic profile. Instead, single- and multiple-turnover experiments gave klim and kmax values identical with kcat values, respectively, indicating that chemistry was now rate-limiting overall. Thus, conserved residues within the steroid-binding pocket affect kcat more than Kd by influencing the rate-determining step of steroid oxidation. These findings support the concept of enzyme catalysis in which the correct positioning of reactants is essential; otherwise, kcat will be limited by the chemical event." @default.
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• W2060603106 date "2004-04-22" @default.
• W2060603106 modified "2023-10-03" @default.
• W2060603106 title "Alanine Scanning Mutagenesis of the Testosterone Binding Site of Rat 3α-Hydroxysteroid Dehydrogenase Demonstrates Contact Residues Influence the Rate-Determining Step" @default.
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• W2060603106 doi "https://doi.org/10.1021/bi0499563" @default.
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