Alanine Scanning Mutagenesis of the Testosterone Binding Site of Rat 3-Hydroxysteroid Dehydrogenase Demonstrates Contact Residues In
详细信息 查看全文
- 作者：Vladi V. Heredia ; William C. Cooper ; Ryan G. Kruger ; Yi Jin ; and Trevor M. Penning
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：May 18, 2004
- 年：2004
- 卷：43
- 期：19
- 页码：5832 - 5841
- 全文大小：320K
- 年卷期：v.43,no.19(May 18, 2004)
- ISSN：1520-4995
文摘
Aldo-keto reductase (AKR1C) isoforms can regulate ligand access to nuclear receptors byacting as hydroxysteroid dehydrogenases. The principles that govern steroid hormone binding and steroidturnover by these enzymes were analyzed using rat 3
-hydroxysteroid dehydrogenase (3-HSD, AKR1C9)as the protein model. Systematic alanine scanning mutagenesis was performed on the substrate-bindingpocket as defined by the crystal structure of the 3-HSD·NADP+·testosterone ternary complex. T24,L54, F118, F129, T226, W227, N306, and Y310 were individually mutated to alanine, while catalyticresidues Y55 and H117 were unaltered. The effects of these mutations on the ordered bi-bi mechanismwere examined. No mutations changed the affinity for NADPH by more than 2-3-fold. Fluorescencetitrations of the energy transfer band of the E·NADPH complex with competitive inhibitors testosteroneand progesterone showed that the largest effect was a 23-fold decrease in the affinity for progesterone inthe W227A mutant. By contrast, changes in the Kd for testosterone were negligible. Examination of thekcat/Km data for these mutants indicated that, irrespective of steroid substrate, the bimolecular rate constantwas more adversely affected when alanine replaced an aromatic hydrophobic residue. By far, the greatesteffects were on kcat (decreases of more than 2 log units), suggesting that the rate-determining step waseither altered or slowed significantly. Single- and multiple-turnover experiments for androsterone oxidationshowed that while the wild-type enzyme demonstrated a klim and burst kinetics consistent with slow productrelease, 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 thatchemistry was now rate-limiting overall. Thus, conserved residues within the steroid-binding pocket affectkcat more than Kd by influencing the rate-determining step of steroid oxidation. These findings support theconcept of enzyme catalysis in which the correct positioning of reactants is essential; otherwise, kcat willbe limited by the chemical event.
-hydroxysteroid dehydrogenase (3-HSD, AKR1C9)as the protein model. Systematic alanine scanning mutagenesis was performed on the substrate-bindingpocket as defined by the crystal structure of the 3-HSD·NADP+·testosterone ternary complex. T24,L54, F118, F129, T226, W227, N306, and Y310 were individually mutated to alanine, while catalyticresidues Y55 and H117 were unaltered. The effects of these mutations on the ordered bi-bi mechanismwere examined. No mutations changed the affinity for NADPH by more than 2-3-fold. Fluorescencetitrations of the energy transfer band of the E·NADPH complex with competitive inhibitors testosteroneand progesterone showed that the largest effect was a 23-fold decrease in the affinity for progesterone inthe W227A mutant. By contrast, changes in the Kd for testosterone were negligible. Examination of thekcat/Km data for these mutants indicated that, irrespective of steroid substrate, the bimolecular rate constantwas more adversely affected when alanine replaced an aromatic hydrophobic residue. By far, the greatesteffects were on kcat (decreases of more than 2 log units), suggesting that the rate-determining step waseither altered or slowed significantly. Single- and multiple-turnover experiments for androsterone oxidationshowed that while the wild-type enzyme demonstrated a klim and burst kinetics consistent with slow productrelease, 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 thatchemistry was now rate-limiting overall. Thus, conserved residues within the steroid-binding pocket affectkcat more than Kd by influencing the rate-determining step of steroid oxidation. These findings support theconcept of enzyme catalysis in which the correct positioning of reactants is essential; otherwise, kcat willbe limited by the chemical event.