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

Abstract

Human type 3 3α-Hydroxysteroid dehydrogenase, or Aldo-Keto Reductase (AKR) 1C2, eliminates the androgen signal in human prostate by reducing 5α-dihydrotesterone (DHT, potent androgen) to form 3α-andostanediol (inactive androgen), thereby depriving the androgen receptor of its ligand. The k cat for the NADPH-dependent reduction of DHT catalyzed AKR1C2 is 0.033 s −1 . We employed transient kinetics and kinetic isotope effects to dissect the contribution of discrete steps to this low k cat 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 k cat (1.5) and a slightly larger effect on k cat /K m (2.1) were observed in the steady state. In the transient state, the maximum rate constant for single turnover of DHT (k trans ) was determined to be 0.11 s −1 for the NADPH-dependent reaction, which was about 4 fold greater than the corresponding k cat . k trans was significantly reduced when NADPD was substituted for NADPH, resulting in an apparent D k trans 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 3-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.Cytosolic hydroxysteroid dehydrogenases (HSDs), which catalyze the NADPH dependent reduction of ketosteroids to hydroxysteroids, are members of the aldo-keto reductase (AKR) superfamily 1C subfamily (1-3). By interconverting potent steroid hormones with their cognate inactive metabolites, AKR1C enzymes can regulate the occupancy of hormone receptors in Address all correspondence to: Dr. Trevor M. Penning, Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084, Phone: 215-898-9445, FAX: 215-573-2236; Email: penning@pharm.med.upenn.edu. 1 Abbreviations and trivial names: HSD, hydroxysteroid dehydrogenase; AKR, aldo-keto reductase; AKR1C2, human type 3 3α-HSD/ bile acid binding protein; DHT, 5α-dihydrotestosterone or 17β-hydroxy-5α-androstan-3-one; Diol, 3α-androstanediol or 5α-androstane-3α,17β-diol; AKR1C9, rat liver 3α-HSD; AKR1B1, human aldose reductase; AKR2B5, yeast xylose reductase; KIE, kinetic isotope effect; 5R2, type 2 5α-reductase. 2 The nomenclature for the aldo-keto reductase superfamily was recommended by the 8 th International Symposium...

Elucidation of a Complete Kinetic Mechanism for a Mammalian Hydroxysteroid Dehydrogenase (HSD) and Identification of All Enzyme Forms on the Reaction Coordinate

“…We have reported a kinetic analysis for the NADPH-dependent reduction of 5␣-DHT catalyzed by AKR1C2, which allows calculation of a K eq of 8.0 for its reduction reaction (39). When combined with the data reported here, it is clear that all AKR1C isoforms will preferentially act as reductases.…”

Oxidation of Dihydrotestosterone by Human Cytochromes P450 19A1 and 3A4

“…Levels of the 19-hydroxy and ⌬ 1,10 -dehydro products did not decrease with time but the 19-aldehyde did. Apparent k cat and K m values were s Ϫ1 reported for reduction of dihydrotestosterone by 3␣-steroid dehydrogenase AKR1C2 (27).…”

“…The enzymes that reduce testosterone to dihydrotestosterone (5␣-steroid reductase, isozymes 1 and 2 (24)) are targets for drugs used to treat prostate hypertrophy, a common problem in older men, as well as some other androgen-dependent conditions (26). The only enzyme reported to be involved in the metabolism of dihydrotestosterone is a 3␣-hydroxysteroid dehydrogenase (AKR1C2) that reduces the 3-keto group to an alcohol, which is devoid of androgen activity (27).…”

An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3α-HSD, type 5 17β-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies

“…AKR1C enzymes catalyze an ordered bi bi mechanism in which cofactor binds first followed by substrate [44][45][46]. Two mechanisms contribute to the inhibition of AKR1C3 by indomethacin and its analogues.…”

“…Inhibitors based on substrates likely favor formation of the E•NADPH•I complexes, while those based on products would be more likely to form E•NADP + •I complexes. With substrates that display high catalytic efficiency, such as PQ [9; 10], the predominate rate-limiting step for reduction is likely NADP + release [49]. This rate determining step will be unaffected by the formation of the E•NADPH•I complex but slowed by the formation of the abortive E•NADP + •I complex, yielding uncompetitive inhibition.…”