Multiple Steps Determine the Overall Rate of the Reduction of 5α-Dihydrotestosterone Catalyzed by Human Type 3 3α-Hydroxysteroid Dehydrogenase: Implications for the Elimination of Androgens

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Abstract
Human type 3 3α-hydroxysteroid dehydrogenase, or aldo-keto reductase (AKR) 1C2, eliminates the androgen signal in human prostate by reducing 5α-dihydrotestosterone (DHT, potent androgen) to form 3α-androstanediol (inactive androgen), thereby depriving the androgen receptor of its ligand. The kcat for the NADPH-dependent reduction of DHT catalyzed by AKR1C2 is 0.033 s-1. We employed transient kinetics and kinetic isotope effects to dissect the contribution of discrete steps to this low kcat value. Stopped-flow experiments to measure the formation of the AKR1C2·NADP(H) binary complex indicated that two slow isomerization events occur to yield a tight complex. A small primary deuterium isotope effect on kcat (1.5) and a slightly larger effect on kcat/Km (2.1) were observed in the steady state. In the transient state, the maximum rate constant for the single turnover of DHT (ktrans) was determined to be 0.11 s-1 for the NADPH-dependent reaction, which was ∼4-fold greater than the corresponding kcat. ktrans was significantly reduced when NADPD was substituted for NADPH, resulting in an apparent Dktrans of 3.5. Thus, the effects of isotopic substitution on the hydride transfer step were masked by slow events that follow or precede the chemical transformation. Transient multiple-turnover reactions generated curvilinear reaction traces, consistent with the product formation and release occurring at comparable rates. Global fitting analysis of the transient kinetic data enabled the estimate of the rate constants for the three-step cofactor binding/release model and for the minimal ordered bi-bi turnover mechanism. Results were consistent with a kinetic mechanism in which a series of slow events, including the chemical step (0.12 s-1), the release of the steroid product (0.081 s-1), and the release of the cofactor product (0.21 s-1), combine to yield the overall observed low turnover number.

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