Contribution of remote substrate binding energy to the enzymatic rate acceleration for 3α-hydroxysteroid dehydrogenase/carbonyl reductase

3 alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-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 3 alpha-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 k(cat), and 37-, 1.9 x 10(6)-, and 1.8 x 10(6)-fold in k(cat)/K-B, respectively. The rate limiting step is hydride transfer for 3 alpha-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 k(cat)/K-B value results in Delta G(double dagger) of 14.7, 12.6, 6.2, and 6.2 kcal/mol for the 3 alpha-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 3 alpha-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 k(cat) for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3 alpha-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 3 alpha-HSD/CR catalysis, resulting in the increases in k(cat) and k(cat)/K-B. (C) 2017 Elsevier B.V. All rights reserved.