title: Contribution of remote substrate binding energy to the enzymatic rate acceleration for 3α-hydroxysteroid dehydrogenase/carbonyl reductase.
abstract: 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidation of androsterone with NAD+to form androstanedione and NADH with the rate limiting step being the release of NADH. In this study, we elucidate the role of remote substrate binding interactions contributing to the rate enhancement by 3α-HSD/CR through steady-state kinetic studies with the truncated substrate analogs. No enzyme activity was detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. As compared to cyclohexanol, the activity for 2-decalol, androstenol, and androsterone increases by 0.9-, 90-, and 200-fold in kcat, and 37-, 1.9 × 106-, and 1.8 × 106-fold in kcat/KB, respectively. The rate limiting step is hydride transfer for 3α-HSD/CR catalyzing the reaction of cyclohexanol with NAD+based on the observed rapid equilibrium ordered mechanism and equal deuterium isotope effects of 3.9 on V and V/K for cyclohexanol. The kcat/KBvalue results in ΔG‡of 14.7, 12.6, 6.2, and 6.2 kcal/mol for the 3α-HSD/CR catalyzed reaction of cyclohexanol, 2-decalol, androstenol, and androsterone, respectively. Thus, the uniform binding energy from the B-ring of steroids with the active site of 3α-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in kcatfor 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3α-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3α-HSD/CR catalysis, resulting in the increases in kcatand kcat/KB.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan. Electronic address: cchwang@kmu.edu.tw.
authors.forename: Chi-Ching
authors.initials: CC
authors.lastname: Hwang
authors.affiliation: Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
authors.forename: Pei-Ru
authors.initials: PR
authors.lastname: Chang
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
authors.forename: Tzu-Pin
authors.initials: TP
authors.lastname: Wang
mesh_terms: D000738:Androsterone / Q000032:analysis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D020604:Comamonas testosteroni / Q000201:enzymology; D003903:Deuterium / Q000737:chemistry; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D009243:NAD; D003903:Deuterium; D000738:Androsterone; C425306:3alpha-hydroxysteroid dehydrogenase-carbonyl reductase; D006913:Hydroxysteroid Dehydrogenases
keywords: 3α-hydroxysteroid dehydrogenase/carbonyl reductase; Binding energy; Enzyme catalysis; Gibbs free energy; Kinetic isotope effect; Steady-state kinetics