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J. Biol. Chem., Vol. 282, Issue 46, 33484-33493, November 16, 2007

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

THE EXAMPLE OF RAT LIVER 3-HSD (AKR1C9)^*

William C. Cooper¹, Yi Jin¹, and Trevor M. Penning²

From the Center of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084

Received for publication, April 24, 2007 , and in revised form, September 7, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP⁺ (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP⁺ controlled k_cat, whereas the chemical event also contributed to this term. k_cat was insensitive to [²H]NADPH, whereas ^Dk_cat/K_m and the ^Dk_lim (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated k_cat, whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall K_eq favored reduction. The kinetic Haldane and free energy diagram confirmed that K_eq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Mammalian hydroxysteroid dehydrogenases (HSDs)³ play pivotal roles in steroid hormone biosynthesis and metabolism (1, 2). In target tissues, they regulate the amount of steroid hormone available for their cognate nuclear receptor (3–7). This is achieved by the positional and stereospecific oxidoreduction of potent steroid hormones to their corresponding inactive metabolites or vice versa. Often these interconversions are catalyzed by pairs of HSDs that function preferentially as reductases or oxidases (5–8). There are two superfamilies of HSDs, the soluble aldo-keto reductase (AKRs) (9) and the mainly membrane-associated short-chain dehydrogenase reductases (SDRs) (10). Several HSDs have become drug targets to manipulate steroid hormone action in target tissues (11–13).

Four human HSDs from the AKR superfamily, AKR1C1–AKR1C4, can reduce 3-, 17-, and 20-ketosteroids in different ratios (14, 15). They catalyze the 4-pro-R hydride transfer from NADPH to the acceptor carbonyl on the steroid substrate in an ordered bi-bi reaction where NAD(P)(H) binds first and leaves last (16, 17). Rat liver 3-HSD (AKR1C9), which shares 69% sequence identity with its human homologs, represents the best system to relate structure to function for HSDs, because crystal structures exist for the apoenzyme (E) (18), the E·NADP⁺ binary complex (19), and the E·NADP⁺·testosterone ternary complex (where testosterone is a competitive inhibitor) (20). These "snapshots" of the enzyme along the reaction pathway reveal that significant conformational changes occur upon the binding of each ligand and provide a structural basis for the ordered bi-bi mechanism (Fig. 1).

Like all AKRs, AKR1C9 adopts an (/)₈-barrel structure that has three large associated loops (loop A–C). In the binary complex, the cofactor is woven through the -helices by 22 contacts with the enzyme. Superimposition of the structures of the apoenzyme and the binary complex showed that the tight binding of the cofactor is associated with significant movement of loops A and B. Interestingly, the ₁-₁ loop and part of loop B, and the C-terminal tail were disordered in the binary complex, but become ordered in the AKR1C9·NADP⁺·testosterone complex to form a mature steroid binding cavity. The influence of loop dynamics on catalysis remains unclear and raises the possibility that additional enzyme forms can exist on the reaction pathway. A complete detailed kinetic mechanism, including microscopic rate constants for conformational changes of all enzyme forms involved in ligand binding, and the chemistry of transformation have yet to be described for any HSD in real-time. Knowledge of the number of enzyme forms that exist is a pre-requisite to identify all the steps that could be intercepted by HSD inhibitors.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Structural changes in AKR1C9 associated with ligand binding revealed by comparing the crystal structures of the apoenzyme E, the E·NADP+ binary complex, and the E·NADP+·testosterone ternary complex. The crystal structures were overlaid to identify conformational changes that occur. Portions of loop structures are colored for the apoenzyme (A), for the binary complex (B), and for the ternary complex (C) according to the following scheme: loop A in cyan, loop B in blue, loop C in orange, and the 1-1 loop in pink. Ligands in the complexes are shown in green (ball and stick presentation). The E·NADP+ binary complex is equivalent to the kinetically observed E**·NADP+, and the E·NADP+·testosterone complex is equivalent to the kinetically observed E***·NADP+·testosterone (see "Results" section).

In this study we elucidate the complete kinetic mechanism for a mammalian HSD at physiological pH using AKR1C9. We find that the reaction sequence is governed by 9 enzyme forms, and 16 kinetic constants were estimated. We also find that the internal equilibrium constant for the reaction favors oxidation but this is not reflected in the overall K_eq, which favors reduction. The kinetic Haldane and the energy diagram confirmed that K_eq was governed by ligand binding terms that favored reactants in the reduction direction. Our findings are discussed within a physiological context.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Materials—Cofactors were purchased from Roche Diagnostics. Steroids were purchased from Steraloids. Deuterated compounds for the synthesis of deuterated NADPH were purchased from Cambridge Isotope Laboratories. NADP⁺-specific alcohol dehydrogenase from Thermoanaerobium brockii was purchased from Sigma. Aldehyde dehydrogenase was purchased from Roche Biochemicals. The expression of pET16b-3-HSD in Escherichia coli DE3 cells and purification of recombinant AKR1C9 have been described previously (25). A specific activity of 1.6 µmol of androsterone oxidized/min/mg of protein was determined. All other reagents were of ACS grade or better. Unless stated otherwise, experiments were carried out at 25 °C in 10 mM potassium phosphate buffer (pH 7.0) and 4% acetonitrile.

Steady-state Kinetic Analysis—Initial velocities were measured on a Hitachi F-4500 fluorescence spectrophotometer by monitoring NADPH depletion or production by the loss or gain of emission at 450 nm upon excitation at 340 nm.

Double reciprocal plots of families of lines in which the initial velocities were plotted against substrate concentrations, androstanedione 0.07–7 µM and NADPH 0.07–8 µM for reduction, and androsterone 0.1–30 µM and NADP⁺ 0.39–27 µM for oxidation, were constructed (26). Data were fitted to the minimal rate equation for an ordered bi-bi reaction, Equation 1 (also see Scheme 1),

(Eq. 1)

with the Cleland FORTRAN program and analyzed for a sequential mechanism. Patterns from plots of families of lines (see "Results") using the SEQUEN program were judged to be the best fit according to the criteria of Cleland (27).

The individual rate constants described in Scheme 1 were obtained using the measured steady-state kinetic parameters based on the definitions of these parameters in terms of rate constants for an ordered bi-bi reaction where the isomerization of the central complex is included in the reaction sequence (28). k_Ab, k_Ar, k_Qr, and k_Qb, which describe cofactor binding and release, were directly calculated. However, the remaining rate constants could only be derived by iteratively fitting six independent algebraic equations for each of the six steady-state parameters that were measured (i.e. k_catRed, K_mNADPH, K_{mAndrostanedione}, k_catOxi, K_mNADP⁺, and K_{mAndrosterone}). Initial estimates and constraints of rate constants were made based on results of transient kinetic experiments. (see "Transient State Kinetic Analysis"). Values of the rate constants were adjusted until all the fits converged simultaneously, i.e. the calculated values of all six steady-state parameters matched the measured values.

View larger version (7K): [in this window] [in a new window]   SCHEME 1. Sequential ordered bi-bi mechanism for AKR1C9.

View larger version (4K): [in this window] [in a new window]   SCHEME 2. Three-step model for NADPH binding to AKR1C9.

Measurement of Equilibrium Constant—The equilibrium constant of the reaction was determined experimentally using a method described by Talalay and Levy (32). In this method, the equilibrium was approached from both the reduction and oxidation directions. Experiments were performed at different cofactor and steroid concentrations. In a typical experiment, the initial reaction mixture contained 4.1 µg of AKR1C9, 22.6 µM androstanedione, 34.6 µM androsterone, and 23 µM NADPH (in 100 mM phosphate buffer, pH 7.0, 5% methanol at 25 °C). When the equilibrium was reached, 8.5 nmol of NADP⁺ was added to force the oxidative reaction and establish a new equilibrium. The changes in absorbance at 340 nm were monitored to calculate the concentrations of reactants and products at each equilibrium end-point permitting the calculation of K_eq.

Transient State Kinetic Analysis—Transient state kinetic assays were performed on an Applied Photophysics SX.18MV-R stopped-flow reaction analyzer instrument fitted with a 20-µl flow cell and a dead time of 1 ms. All concentrations are given as final.

Kinetics of Binary Complex Formation—The binding of NADPH to AKR1C9 was monitored in real-time using stopped-flow spectroscopy by exciting the protein at 290 nm and capturing the emission of the energy transfer band at 450 nm through a 10-nm band-pass filter. The energy transfer band is produced as a result of the interaction of NADPH with Trp-86 (33). Conversely, to monitor the binding of NADP⁺, the decrease in protein fluorescence emission at 330 nm was recorded upon quenching the excited tryptophanyl residues at 290 nm. In a typical experiment, AKR1C9 (0.25 µM) was mixed with NADPH (2.0–11 µM). For NADP⁺ binding, enzyme (0.70 µM) was mixed with NADP⁺ (4.4–36 µM). The time courses of the fluorescence increase or decrease at each concentration of ligand were best fitted to double exponential equation (Equation 2),

(Eq. 2)

where F_t is the fluorescence at time t, F is the amplitude of the fluorescence increase or decrease, k_obs1 and k_obs2 are the apparent first-order rate constants for the fast and slow phases, respectively, and F_eq is the fluorescence at equilibrium. Data were analyzed based on a three-step model for cofactor binding (Scheme 2). Estimates of the kinetic constants K_{1 NADPH} and k₃–k₅ were obtained by the method of Stone and Le Bonniec (34). K_{1 NADPH} is the apparent dissociation constant of the initial loose complex. The plot of k_obs2 versus [NADPH] provided the estimate of k₅. Values of K_dNADPH were determined by steady-state fluorescence titration (25), and estimates of K₁ and k₃–k₅ were substituted into Equation 3 to solve for k₆.

(Eq. 3)

Similarly, estimates of kinetic constants for the binding of NADP⁺ (k₁₃–k₁₆ and K_1NADP⁺) were obtained.

Ligand Chase Experiments—Enzyme (0.4 µM) was incubated with NADPH (12 µM) in one syringe and mixed with NADP⁺ (40 µM) from a second syringe. The decrease of the energy transfer band corresponding to dissociation of NADPH from the binary complex was monitored at 450 nm. Transients were best fitted to a bi-exponential (Equation 2).

Kinetics of Ternary Complex Formation—A solution containing enzyme (0.4 µM) and NADPH (12 µM) from one syringe was mixed with increasing concentrations of testosterone (3.60–6.15 µM) from a second syringe. Decreases in fluorescence at 450 nm were monitored with excitation at 295 nm. Fluorescence traces were marked by a single exponential decay. Five transient traces were averaged for each steroid concentration, and values for k_obs were fitted to Equation 4 to solve for k_on and k_off (35).

(Eq. 4)

Transient State Turnover Experiments—For single turnover experiments, AKR1C9 (0.5 µM) was incubated with substoichiometric amounts of NADPH (0.3 µM) in one syringe and mixed with a varying excess of androstanedione (4–15.0 µM) added from a second syringe in the stopped-flow instrument. Similarly for single turnover of androsterone oxidation, a solution of AKR1C9 (0.8 µM) and NADP⁺ (0.6 µM) from one syringe were mixed with an excess of androsterone ranging from 7.0 to 30 µM from a second syringe. Reactions were monitored fluorimetrically (excitation at 340 nm and emission at 450 nm). Data were fitted to a single exponential equation. Plots of k_obs versus steroid concentration were fitted to the hyperbolic equation (Equation 5), where A is the steroid concentration, k_lim is the maximum rate of single turnover, and K_d is a quasi dissociation constant for steroid substrate (34).

(Eq. 5)

Values of k_lim provided the lower limits for the k_+p and k_-p and initial constraints on k_Bb, k_Br, k_Pr, and k_Pb were set based on values of K_d during steady-state fitting analysis.

To study multiple turnover of androstanedione reduction, a solution of AKR1C9 (1.5 µM) and NADPH (25 µM) was mixed with androstanedione (13.5 µM). A complete concentration series with androstanedione was not possible due to steroid solubility constraints. Kinetic transients of NADPH depletion at 450 nm were collected. Experiments were repeated in the absorbance mode. Similarly, multiple turnover experiments of androsterone oxidation were conducted in which AKR1C9 (1 µM) and NADP⁺ (40 µM) from one syringe were mixed with increasing concentrations of androsterone (10–35 µM) added from a second syringe. Transient traces were fitted to an equation (Equation 6) that contained both burst and steady-state terms,

(Eq. 6)

where F_t equals fluorescence at time t, Amp equals the amplitude of the burst, k_obs is the apparent rate constant of the burst in units of s^-1, v_ss is the steady-state rate in units of F/t, and c is the fluorescence at equilibrium (35). The maximum value of k_obs yielded k_burst, and the maximum value of v_ss was, subsequently, mathematically transformed into micromolar/second and divided by enzyme concentration to yield k_ss (s^-1).

Transient state single and multiple turnover primary KIEs were determined by substituting NADPD in single turnover and multiple turnover experiments. ^Dk_lim is the ratio of k_lim determined in the presence of NADPH versus NADPD, and ^Dk_lim/K_d is the ratio of k_lim/K_d determined in the presence of NADPH versus NADPD. k_lim and K_d are given by Equation 5.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Dissection of the complete kinetic mechanism for AKR1C9 was conducted using androstanedione and NADPH, and androsterone and NADP⁺, as substrate pairs at physiological pH. Androstanedione and androsterone were chosen, because these steroids have been standards with which all other assays on this enzyme in our laboratory have been compared. NADP(H) cofactors were chosen, because bound NADP⁺ was present in the crystal structures of the binary and ternary complexes. Thus, the existence of kinetically inferred complexes could be related to structural changes in the enzyme.

Steady-state Initial Velocity Studies—Initial velocity studies for androstanedione reduction and androsterone oxidation using NADP(H) were performed. Double reciprocal plots of the initial velocity data (see supplemental data) were characterized by a fan of lines that converged to an intersecting point to the left of the origin and above or on the x-axis. This is indicative of a sequential mechanism defined by the requirement of the cofactor and substrate to bind to the enzyme to form a ternary complex before any product can be produced (28). The sequential mechanism was previously shown to be an ordered bi-bi reaction as seen in Scheme 1. Fits of family of lines were generated through the Cleland SEQUEN program to yield steady-state kinetic parameters given in Table 1.

(Eq. 7)

The steady-state parameters in Table 1 gave a value of 22.7 for this expression where the forward direction is reduction. This is in good agreement with a value of 20.9 ± 1.8 determined experimentally using a method described by Talalay and Levy (32). The kinetic Haldane revealed that the K_eq is dominated by submicromolar K_i and K_m values for NADPH and androstanedione, respectively.

Rate Constants Obtained in the Steady State—The individual rate constants described in Scheme 1 were calculated and derived from the measured steady-state parameters (Table 2). In the reduction direction these constants showed that the binding of NADPH (k_Ab) was slow and did not occur at the diffusion limit. The binding of androstanedione (k_Bb) to the binary complex occurred 5-times faster than k_Ab, and the rate of conversion of the central complex (k_+p) was 6.6 s^-1. The release of the steroid product androsterone (k_Pr) occurred at a comparable rate of 10.5 s^-1. The main contribution to the overall k_cat of 0.42 s^-1 came from the release of the NADP⁺ product, k_Qr = 0.65 s^-1.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Fluorescence kinetic transients for the binding of NADP+ to AKR1C9. A, a representative fluorescence transient trace for the binding of NADP+ (2.5 µM) to AKR1C9 (0.25 µM) fitted to a mono-exponential function; B, the same trace in A fitted to a bi-exponential function; The residuals are shown under A and B; C, concentration dependence of kobs (s-1) from the fast phase (•) and slow phase () of the bi-exponential fittings.

The kinetic rate constants defined in Scheme 2 were estimated using the transient data and are listed in Table 3. The dissociation constants for the initial loose complexes were determined to be 12 µM and 35 µM for E·NADPH and E·NADP⁺, respectively. Compared with the apparent K_d values determined experimentally by fluorescence titration, the sequential conformational change steps resulted in 86- and 110-fold increases in affinity for NADPH and NADP⁺ binding, respectively.

Kinetics of Ketosteroid Binding—Because it is not possible to directly measure the kinetics of the binding of 3-ketosteroid steroid substrates we conducted experiments with testosterone, a competitive inhibitor and related 3-ketosteroid. Testosterone is also the ligand present in the crystal structure of the ternary complex. Quenching of the energy transfer band upon testosterone binding to the enzyme NADPH binary complex was marked by a decrease in fluorescence at 450 nm in the steady state and yielded a K_d value of 0.5 µM obtained by fluorescence titration.⁴ Stopped-flow experiments were then performed to determine whether steroid binding was accompanied with a fluorescence kinetic transient. Each transient trace was fitted to a single exponential equation (Fig. 3). At high testosterone concentrations the amplitude decayed too rapidly and the observable rate constant could not be discerned. As a result, saturation kinetics was not detected. At lower steroid concentrations, the concentration dependence on k_obs was linear. The microscopic association and dissociation rate constants were estimated to be 95.7 ± 4.8 µM^-1 s^-1 and 14.1 ± 1.9 s^-1, respectively, and gave a K_d of 0.15 µM.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Transient kinetics of testosterone binding. A, a representative fluorescence transient trace for the quenching of the energy transfer band at 450 nm upon mixing a preincubated solution of AKR1C9 (0.4 µM) and saturating NADPH (12 µM) with testosterone (3.6 µM); B, the dependence of kobs on varying testosterone concentrations.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Reduction of androstanedione by NADPH or NADPD and oxidation of androsterone by NADP+ under single turnover conditions. A, a representative fluorescence transient trace at 450 nm for the single turnover of androstanedione reduction obtained by mixing a solution of AKR1C9 (0.5 µM) and NADPH (lower trace) or NADPD (upper trace) (0.3 µM) with androstanedione (10 µM). The transient was fitted to a mono-exponential function to obtain kobs; B, the dependence of kobs on androstanedione concentration with NADPH (•) and NADPD (); C, a representative transient kinetic trace for the single turnover of androsterone oxidation obtained by mixing a solution of enzyme (0.8 µM) and cofactor (0.6 µM) with androsterone (20 µM). The transient was fitted to a mono-exponential function to obtain kobs; D, dependence of kobs on substrate concentration for androsterone oxidation.

Transient-state multiple turnover experiments were also performed to determine whether androsterone oxidation was accompanied by burst-phase kinetics. Androsterone oxidation was characterized by a pronounced burst that yielded a k_burst (55.8 s^-1) that was similar to k_lim (35.7 s^-1) (Fig. 5C). The amplitude of the burst coincided with the concentration of the enzyme. The k_ss (0.77 s^-1) from androsterone oxidation under multiple turnovers was in accord with k_cat (0.82 s^-1).

Primary KIEs on Steady-state and Transient State Kinetic Parameters—To confirm that the chemical step was not rate-determining for androstanedione reduction, primary KIE values were performed using NADPD. There was no significant ^Dk_cat nor ^Dk_cat/K_m, and values of 1.08 and 1.06 were obtained (Table 4). By contrast under single turnover conditions, ^Dk_lim = 2.06 was observed (Fig. 4). Under multiple turnover conditions a ^Dk_burst equal to 2.07 was noted and was reduced to 0.93 for Dk_ss (Fig. 5A). The data were internally consistent with steps other than chemistry being rate-determining in the reduction of androstanedione.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
A complete kinetic mechanism for a mammalian HSD has been presented (see Table 3 and Scheme 3). The mechanism contains 9 enzyme forms, and 16 individual kinetic constants were assigned. The steady-state kinetic parameters for the mechanism were reconciled with rate constants obtained from the transient-state kinetic measurements. This mechanism can be used as a guide for studies on other HSDs.

Rate-determining Steps in AKR1C9 Catalysis—Our study focused on the NADP(H)-dependent interconversion of androstanedione and androsterone catalyzed by AKR1C9 using steady-state and transient-state kinetics. The contributions of different steps to rate determination and hence k_cat can be dissected with Equations 8, 9, 10, 11. In the reduction direction, the release of NADP⁺ (0.65 s^-1) largely (64%) controls the k_cat of 0.42 s^-1. However, the net rate for the chemical reduction of androstanedione (k_chemRed of 1.3 s^-1) also contributed significantly (33%), and this was due to the size of k_-p for the oxidation reaction in the steady state. The small difference between k_chemRed and the net rate of the subsequent product release steps (0.61 s^-1) resulted in the partial burst seen under multiple turnover conditions. In contrast, the k_cat of 0.82 s^-1 for the oxidation direction was dominated (96%) by the release of NADPH (0.85 s^-1). The 30-fold greater k_chemOxi (24.2 s^-1 caused the full burst observed under multiple turnover conditions for the oxidation reaction.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Reduction of androstanedione by NADPH or NADPD and oxidation of androsterone by NADP+ under multiple turnover conditions. A, a premixed solution of AKR1C9 (1.5 µM) and NADPD (upper trace) or NADPH (lower trace) (25 µM) was mixed with androstanedione (13.5 µM). Product formation corresponding to the decrease in fluorescence emission at 450 nm was monitored; B, a representative transient trace obtained in the absorbance mode where a solution of enzyme (2 µM) and NADPH (30 µM) were mixed with androstanedione (16 µM). The decrease in absorbance at 340 nm was monitored. C, a representative transient kinetic trace for androsterone oxidation under multiple turnover conditions where AKR1C9 (1 µM) and NADP+ (40 µM) were mixed with androsterone (15 µM).

(Eq. 8)

(Eq. 9)

(Eq. 10)

(Eq. 11)

We have previously shown that the NADPH reduction of 5-DHT catalyzed by AKR1C9 had a distinctively different kinetic profile (36). In this reaction, there was no burst-phase kinetics in multiple turnover experiments, and a presence of significant primary KIEs provided evidence that chemistry was a major rate-determining step. Thus AKR1C9 can catalyze identical reactions on two highly related 3-ketosteroids (5-DHT and androstanedione) with different rate-determining profiles. This is likely due to the different positioning of the steroid at the active site and changes in proximity between the reactants. We have previously shown using alanine-scanning mutagenesis that subtle changes in the binding of steroids to AKR1C9 can alter the rate-determining step of the reaction (37).

View larger version (12K): [in this window] [in a new window]   SCHEME 3. Complete kinetic mechanism for AKR1C9.

To progress from the binary complex to the ternary complex, structural data imply that additional ordering of the loops occurs (19). Kinetic transients were captured possibly reflecting these changes for the binding of testosterone to AKR1C9. The microscopic association and dissociation rate constants for testosterone binding were estimated to be 95.7 µM^-1s^-1 and 14.1 s^-1 and approached those for androstanedione obtained in the steady-state solution, which were 55.2 µM^-1s^-1 and 27.8 s^-1. Significantly, these values differ from the association and dissociation rate constants for androsterone obtained from steady-state analysis, which were 0.26 µM^-1s^-1 and 10.5 s^-1, respectively. This reflects a 80-fold decrease in the K_d value for androstanedione. Considering the structural differences between androstanedione (substrate) and androsterone (product) in the reduction direction, it is apparent that the enzyme has evolved to favor 3-ketosteroid binding, i.e. the binding of reductive substrates.

Equilibrium Constants and Free Energy Diagram—The concept that HSDs work in pairs to act as either reductases or oxidases and thereby regulate ligand access to steroid hormone receptors is based to a large extent on transfection assays using radiotracers. In transfection paradigms HSDs are forced to use the prevailing concentrations of reduced or oxidized cofactor that exist in the recipient cell line. By contrast HSDs in vitro can be bi-directional providing that the NAD(P)(H) concentration is manipulated to drive the reaction in the desired direction. There have been few attempts to reconcile these disparate findings with the K_eq of these reactions. In this study we measured a K_eq of 20.9 for the NADPH-dependent reduction of androstanedione, which is in close agreement with that determined by the kinetic Haldane, which gave a value of 22.7. These values also agree with the K_eq of 14.4 for the NADPH-dependent reduction of 5-DHT catalyzed by AKR1C9 (36) and the K_eq of 12.0 for the NADH-dependent reduction of androstanedione catalyzed by AKR1C9 (17). Thus, irrespective of the cofactor pair, AKR1C9 behaves preferentially as a reductase.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Free energy diagram for the NADPH-dependent reduction of androstanedione catalyzed by AKR1C9. Changes of the Gibbs free energy associated with each step described in Scheme 1 for the NADP(H)-dependent interconversion of androstanedione and androsterone were calculated according to Equations 12 and 13.

(Eq. 12)

(Eq. 13)

where k_f and k_r are the forward and reverse rate constants of the step, respectively, R is the gas constant, T is the absolute temperature, and k_B and h are the Boltzmann and Planck constants, respectively. The free-energy profile was similar for both directions. In the reduction direction, the binding of NADPH and androstanedione are associated with free energy changes (₁ and ₂) of -9.2 and -7.8 kcal/mol, respectively. However, the free-energy change for the chemical conversion (₃) is only +1.1 kcal/mol, while the energy required to reach the transition state for the reaction (₃) was +16.3 kcal/mole. The subsequent gain in free energy for the product releases steps were ₄ of +6.0 kcal/mol and ₅ of +8.1 kcal/mol.

The overall free-energy change (_total) for the reaction is -1.8 kcal/mol, indicating that the reduction is favored thermodynamically. However, the internal K_eq based on single turnover k_limRed:k_limOxi (0.13) or the steady-state solution k_+p:k_-p (0.15) indicates that the oxidation reaction is favored. By contrast, the K_eq for the entire action is 22.7 and is 170 times larger than the internal K_eq. The reason for this large change can be accounted for by ligand binding terms, which favor NADPH and androstanedione. In comparison to other members of the AKR superfamily, the K_eq of AKR1C9 is low. Thus aldose reductase, a prototypical AKR, has a K_eq of 150 in favor of reduction (38). The K_eq values and internal K_eq values for other mammalian HSDs, including the SDRs, have yet to be reported.

Physiological Significance—In humans four highly related AKR1C isoforms exist (AKR1C1–AKR1C4). 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. Thus the human enzymes will function as 3-, 17-, and 20-ketoreductases. The unexpected finding of this study is the K_eq does not appear to be governed by cofactor, because the same K_eq is observed irrespective of whether reduction is coupled to NADPH/NADP⁺ or NADH/NAD⁺. Rather, the K_eq is governed by favorable binding terms for the reduction reactants. Also, the K_eq is not overwhelmingly in favor of reduction. The K_eq values reported are rather modest and indicate that under some conditions the oxidative reaction may be observed. Two observations suggest that this can happen. Transfection of AKR1C9 into HEK293 cells showed that a pseudo-equilibrium was reached in which the product to substrate ratio was 97:3 (24). At this equilibrium the forward and reverse reaction rates were identical. Second, in studies on the metabolism of 3-hydroxysteroids in human hepatoma cells we noted that, although the favored metabolic route was formation of steroid conjugates, a small amount of epimerization (<5%) to the 3-hydroxysteroid metabolites was observed that could only be explained by oxidation back to the 3-ketosteroid (40).

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
AKR1C9, an HSD, is a member of the steroid hormone-transforming AKRs that regulate ligand occupancy of steroid receptors. Understanding the number of enzyme forms and their effects on rate determination of steroid transformation identifies steps for interception by inhibitors and regulation of these reactions. This study revealed that in the reaction sequence there are nine enzyme forms and six contain NAD(P)H. It also showed that, although the internal K_eq of AKR1C9 clearly favors oxidation, which is unique among AKRs, the overall K_eq favors reduction. This difference in AKRs is achieved by slow product release steps that control the reaction in both directions. The K_eq that favors reduction is dominated by ligand binding terms for the reduction reactants.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants R01-DK47015 and P30 ES013508 (to T. M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Pharmacology, University of Pennsylvania, 130C John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Tel.: 215-573-2236; Fax: 215-573-2236; E-mail: penning{at}pharm.med.upenn.edu.

³ The abbreviations used are: HSD, hydroxysteroid dehydrogenase; AKR, aldoketo reductase; AKR1C2, human type 3 3-HSD; AKR1C9, rat liver 3-HSD; androstanedione, androstane-3,17-dione; androsterone, 17-hydroxy-5-androstane-3-one; 5-DHT, 5-dihydrotestosterone; KIE, kinetic isotope effect; SDR, short-chain dehydrogenase reductase; NADPD, [²H]NADPH.

⁴ W. C. Cooper, unpublished results.

	ACKNOWLEDGMENTS

 
We thank Jason E. Drury and Carol A. Shultz for providing purified AKR1C9 and Vladi V. Heredia for helpful discussions.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 

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