Elucidation of a Complete Kinetic Mechanism for a Mammalian Hydroxysteroid Dehydrogenase (HSD) and Identification of All Enzyme Forms on the Reaction Coordinate
2007; Elsevier BV; Volume: 282; Issue: 46 Linguagem: Inglês
10.1074/jbc.m703414200
ISSN1083-351X
AutoresWilliam C. Cooper, Yi Jin, T.M. Penning,
Tópico(s)Coenzyme Q10 studies and effects
ResumoHydroxysteroid 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 3α-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 kcat, whereas the chemical event also contributed to this term. kcat was insensitive to [2H]NADPH, whereas Dkcat/Km and the Dklim (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 kcat, 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 Keq favored reduction. The kinetic Haldane and free energy diagram confirmed that Keq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction. 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 3α-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 kcat, whereas the chemical event also contributed to this term. kcat was insensitive to [2H]NADPH, whereas Dkcat/Km and the Dklim (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 kcat, 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 Keq favored reduction. The kinetic Haldane and free energy diagram confirmed that Keq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction. Mammalian hydroxysteroid dehydrogenases (HSDs) 3The abbreviations used are: HSD, hydroxysteroid dehydrogenase; AKR, aldoketo reductase; AKR1C2, human type 3 3α-HSD; AKR1C9, rat liver 3α-HSD; androstanedione, androstane-3,17-dione; androsterone, 17β-hydroxy-5α-androstane-3-one; 5α-DHT, 5α-dihydrotestosterone; KIE, kinetic isotope effect; SDR, short-chain dehydrogenase reductase; NADPD, [2H]NADPH. play pivotal roles in steroid hormone biosynthesis and metabolism (1Penning T.M. Endocr. Rev. 1997; 18: 281-305Crossref PubMed Scopus (391) Google Scholar, 2Payne A.H. Hales D.B. Endocr. Rev. 2004; 25: 947-970Crossref PubMed Scopus (1207) Google Scholar). In target tissues, they regulate the amount of steroid hormone available for their cognate nuclear receptor (3Funder J.W. Pearce P.T. Smith R. Smith A.I. Science. 1988; 242: 583-585Crossref PubMed Scopus (1484) Google Scholar, 4Draper N. Stewart P.M. J. Endocrinol. 2005; 186: 251-271Crossref PubMed Scopus (318) Google Scholar, 5Rižner T.L. Lin H.-K. Peehl D.M. Steckelbroeck S. Bauman D.R. Penning T.M. Endocrinology. 2003; 144: 2922-2932Crossref PubMed Scopus (122) Google Scholar, 6Bauman D.R. Steckelbroeck S. Williams M.V. Peehl D.M. Penning T.M. Mol. Endocrinol. 2006; 20: 444-458Crossref PubMed Scopus (104) Google Scholar, 7Penning T.M. Bauman D.R. Jin Y. Rižner T.L. Mol. Cell. Endocrinol. 2007; 265-266: 77-82Crossref PubMed Scopus (43) Google Scholar). 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 (5Rižner T.L. Lin H.-K. Peehl D.M. Steckelbroeck S. Bauman D.R. Penning T.M. Endocrinology. 2003; 144: 2922-2932Crossref PubMed Scopus (122) Google Scholar, 6Bauman D.R. Steckelbroeck S. Williams M.V. Peehl D.M. Penning T.M. Mol. 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Norstrom C. Stefansson K. Abrahmsen L. Oppermann U. Svensson S. J. Biol. Chem. 2005; 280: 3789-3794Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13Hosfield D.J. Wu Y. Skene R.J. Hilgers M. Jennings A. Snell G.P. Aertgeerts K. J. Biol. Chem. 2005; 280: 4639-4648Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Four human HSDs from the AKR superfamily, AKR1C1–AKR1C4, can reduce 3-, 17-, and 20-ketosteroids in different ratios (14Penning T.M. Burczynski M.E. Jez J.M. Hung C.-F. Lin H.-K. Ma H. Moore M. Palackal N. Ratnam K. Biochem. J. 2000; 351: 67-77Crossref PubMed Scopus (540) Google Scholar, 15Steckelbroeck S. Jin Y. Gopishetty S. Oyesanmi B. Penning T.M. J. Biol. Chem. 2004; 279: 10784-10795Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). 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 (16Ricigliano J.W. Penning T.M. Biochem. J. 1990; 269: 749-755Crossref PubMed Scopus (10) Google Scholar, 17Askonas L.J. Ricigliano J.W. Penning T.M. Biochem. J. 1991; 278: 835-841Crossref PubMed Scopus (68) Google Scholar). Rat liver 3α-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) (18Hoog S.S. Pawlowski J.E. Alzari P.M. Penning T.M. Lewis M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2517-2521Crossref PubMed Scopus (141) Google Scholar), the E·NADP+ binary complex (19Bennett M.J. Schlegel B.P. Jez J.M. Penning T.M. Lewis M. Biochemistry. 1996; 35: 10702-10711Crossref PubMed Scopus (104) Google Scholar), and the E·NADP+·testosterone ternary complex (where testosterone is a competitive inhibitor) (20Bennett M.J. Albert R.H. Jez J.M. Ma H. Penning T.M. Lewis M. Steroids. 1997; 5: 799-812Google Scholar). 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).TABLE 1Steady-state kinetic parameters for androstanedione reduction and androsterone oxidation catalyzed by AKR1C9ParameterMeasuredCalculatedaSteady-state parameters were calculated with the equations that define the steady state kinetic parameters in terms of individual rate constants where the interconversion of the central complex is taken into account (28) using the estimates of rate constants listed in Table 2ParameterMeasuredCalculatedaSteady-state parameters were calculated with the equations that define the steady state kinetic parameters in terms of individual rate constants where the interconversion of the central complex is taken into account (28) using the estimates of rate constants listed in Table 2kcatRed (s-1)0.42 ± 0.020.41kcatOxi (s-1)0.82 ± 0.040.82KmNADPH (μm)0.083 ± 0.0030.081KmNADP+ (μm)1.51 ± 0.21.53KmAdionebAdione = androstanedione and Arone = androsterone (μm)0.65 ± 0.040.65KmAronebAdione = androstanedione and Arone = androsterone (μm)4.05 ± 0.54.05KiNADPH (μm)0.17 ± 0.040.17KiNADP+ (μm)1.20 ± 0.361.20a Steady-state parameters were calculated with the equations that define the steady state kinetic parameters in terms of individual rate constants where the interconversion of the central complex is taken into account (28Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley & Sons, New York1993: 274-574Google Scholar) using the estimates of rate constants listed in Table 2b Adione = androstanedione and Arone = androsterone Open table in a new tab SCHEME 1Sequential ordered bi-bi mechanism for AKR1C9.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 1Structural 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 β1-α1 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Like all AKRs, AKR1C9 adopts an (α/β)8-barrel structure that has three large associated loops (loop A–C). In the binary complex, the cofactor is woven through the α-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 β1-α1 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. The hydride transfer reaction catalyzed by HSDs is reversible in vitro. The seemingly directional preference of an individual HSD, either an AKR or an SDR, seen in transfection studies is believed to be governed by the cofactor ratio and cofactor preference. In the cytosol, the relationships are [NADPH] >> [NADP+] and [NAD+] >> [NADH] (21Veech R.L. Eggleston I.V. Krebs H.A. Biochem. J. 1969; 115: 609-619Crossref PubMed Scopus (426) Google Scholar, 22Williamson D.H. Lund P. Krebs H.A. Biochem. J. 1967; 103: 514-527Crossref PubMed Scopus (1308) Google Scholar). Thus enzymes that have high affinity for NADPH will act as reductases, and those that preferentially utilize NADH/NAD will act as oxidases. AKRs have nanomolar affinity for the NADPH/NADP+ pair. We found that the NADPH-dependent reduction of 5α-DHT catalyzed by human type 3 3α-HSD (AKR1C2) occurred unimpeded in the presence of 1 mm NAD+ but that the NAD+-dependent oxidation of 3α-androstanediol oxidation catalyzed by AKR1C2 was potently inhibited by low micromolar concentrations of NADPH, suggesting that the reaction is unidirectional under normal cellular redox conditions (5Rižner T.L. Lin H.-K. Peehl D.M. Steckelbroeck S. Bauman D.R. Penning T.M. Endocrinology. 2003; 144: 2922-2932Crossref PubMed Scopus (122) Google Scholar). Recently, transfection studies with HSDs showed that a functional pseudo-equilibrium was reached such that the rates of the forward and reverse reactions were identical (23Khun N. Sharma K.K. Andersson S. Auchus R.J. Arch. Biochem. Biophys. 2004; 429: 50-59Crossref PubMed Scopus (46) Google Scholar, 24Papari-Zareei M. Brandmaier A. Auchus R.J. Endocrinology. 2006; 147: 1591-1597Crossref PubMed Scopus (20) Google Scholar). Thus, directionality and/or reversibility of an HSD are also related to the equilibrium constant of the reaction, which has generally not been reported for mammalian HSDs. 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 Keq, which favors reduction. The kinetic Haldane and the energy diagram confirmed that Keq was governed by ligand binding terms that favored reactants in the reduction direction. Our findings are discussed within a physiological context. 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-3α-HSD in Escherichia coli DE3 cells and purification of recombinant AKR1C9 have been described previously (25Ratnam K. Ma H. Penning T.M. Biochemistry. 1999; 38: 7856-7864Crossref PubMed Scopus (68) Google Scholar). 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 (26Cleland W.W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (1930) Google Scholar). Data were fitted to the minimal rate equation for an ordered bi-bi reaction, Equation 1 (also see Scheme 1), v=VmAB/(KiAKmB+KmAB+KmBA+AB)(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 (27Cleland W.W. Adv. in Enzymol. 1977; 45: 273-387Google Scholar). 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 (28Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley & Sons, New York1993: 274-574Google Scholar). kAb, kAr, kQr, and kQb, 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. kcatRed, KmNADPH, KmAndrostanedione, kcatOxi, KmNADP+, and KmAndrosterone). 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. The synthesis of 4-pro-R-[2H]NADPH (NADPD) was performed as described by Hermes et al. (29Hermes J.D. Morrical S.W. O'Leary M.H. Cleland W.W. Biochemistry. 1984; 23: 5479-5488Crossref PubMed Scopus (133) Google Scholar) and Viola et al. (30Viola R.E. Cook P.F. Cleland W.W. Anal. Biochem. 1979; 96: 334-340Crossref PubMed Scopus (135) Google Scholar) with minor modifications. Analysis of the NADPD product and assessment of deuterium incorporation was determined at 2.9 ppm by [1H]NMR. The [2H] content at the 4-pro-R position was estimated to be >99%. For estimates of primary KIEs, initial velocities were performed as described previously by substituting NADPD in the assays. The nomenclature of Northrop (31Northrop D.B. Methods Enzymol. 1982; 87: 607-625Crossref PubMed Scopus (108) Google Scholar) was used where Dkcat is the ratio of kcat determined in the presence of NADPH versus NADPD, and Dkcat/Km is the ratio of kcat/Km determined in the presence of NADPH versus NADPD. Measurement of Equilibrium Constant—The equilibrium constant of the reaction was determined experimentally using a method described by Talalay and Levy (32Talalay P. Levy H.R. Ciba Foundation Study Group No. 2: Steric Course of Microbiological Reactions. Little, Brown & Co, Boston1959: 64-66Google Scholar). 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 Keq. 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 (33Jez J.M. Schlegel B.P. Penning T.M. J. Biol. Chem. 1996; 271: 30190-30198Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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), Ft=(ΔF) 1e−kobs1t+(ΔF) 2e−kobs2t+Feq(Eq. 2) where Ft is the fluorescence at time t, ΔF is the amplitude of the fluorescence increase or decrease, kobs1 and kobs2 are the apparent first-order rate constants for the fast and slow phases, respectively, and Feq is the fluorescence at equilibrium. Data were analyzed based on a three-step model for cofactor binding (Scheme 2). Estimates of the kinetic constants K1 NADPH and k3–k5 were obtained by the method of Stone and Le Bonniec (34Stone S.R. Le Bonniec B.F. J. Mol. Biol. 1997; 265: 344-362Crossref PubMed Scopus (51) Google Scholar). K1 NADPH is the apparent dissociation constant of the initial loose complex. The plot of kobs2 versus [NADPH] provided the estimate of k5. Values of KdNADPH were determined by steady-state fluorescence titration (25Ratnam K. Ma H. Penning T.M. Biochemistry. 1999; 38: 7856-7864Crossref PubMed Scopus (68) Google Scholar), and estimates of K1 and k3–k5 were substituted into Equation 3 to solve for k6. Kd=K1NADPHK4K6/(K3K5+K4K6+K3K6)(Eq. 3) Similarly, estimates of kinetic constants for the binding of NADP+ (k13–k16 and K1NADP+) 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 kobs were fitted to Equation 4 to solve for kon and koff (35Johnson K.A. The Enzymes. 1992; 20: 1-62Crossref Scopus (378) Google Scholar). Kobs=Kon[T]+Koff(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 kobs versus steroid concentration were fitted to the hyperbolic equation (Equation 5), where A is the steroid concentration, klim is the maximum rate of single turnover, and Kd is a quasi dissociation constant for steroid substrate (34Stone S.R. Le Bonniec B.F. J. Mol. Biol. 1997; 265: 344-362Crossref PubMed Scopus (51) Google Scholar). Kobs=Klim[A]/(Kd+[A])(Eq. 5) Values of klim provided the lower limits for the k+p and k-p and initial constraints on kBb, kBr, kPr, and kPb were set based on values of Kd 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, Ft=Ampe−kobst+vsst+c(Eq. 6) where Ft equals fluorescence at time t, Amp equals the amplitude of the burst, kobs is the apparent rate constant of the burst in units of s-1, vss is the steady-state rate in units of ΔF/Δt, and c is the fluorescence at equilibrium (35Johnson K.A. The Enzymes. 1992; 20: 1-62Crossref Scopus (378) Google Scholar). The maximum value of kobs yielded kburst, and the maximum value of vss was, subsequently, mathematically transformed into micromolar/second and divided by enzyme concentration to yield kss (s-1). Transient state single and multiple turnover primary KIEs were determined by substituting NADPD in single turnover and multiple turnover experiments. Dklim is the ratio of klim determined in the presence of NADPH versus NADPD, and Dklim/Kd is the ratio of klim/Kd determined in the presence of NADPH versus NADPD. klim and Kd are given by Equation 5. 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 (28Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley & Sons, New York1993: 274-574Google Scholar). 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. It was found that kcat = 0.82 s-1 for the oxidation direction was 2-fold greater than the reduction direction, kcat = 0.42 s-1, whereas KmNADPH was smaller than KmNADP+ by > 18-fold. Ki values for the cofactors were determined to be 0.17 μm and 1.51 μm for NADPH and NADP+, respectively, and were in reasonable agreement with the Kd values of 0.14 μm and 0.32 μm determined directly by fluorescence titration (25Ratnam K. Ma H. Penning T.M. Biochemistry. 1999; 38: 7856-7864Crossref PubMed Scopus (68) Google Scholar). The internal consistency of the kinetic parameters in Table 1 was checked with the kinetic Haldane relationship for an ordered bi-bi mechanism (28Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley & Sons, New York1993: 274-574Google Scholar) by Equation 7. Keq=VmaxRedKiNADP+KmAndrosterone/(VmaxOxiKiNADPHKmAndrostanedione)(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 (32Talalay P. Levy H.R. Ciba Foundation Study Group No. 2: Steric Course of Microbiological Reactions. Little, Brown & Co, Boston1959: 64-66Google Scholar). The kinetic Haldane revealed that the Keq is dominated by submicromolar Ki and Km 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 (kAb) was slow and did not occur at the diffusion limit. The binding of androstanedione (kBb) to the binary complex occurred 5-times faster than kAb, and the rate of conversion of the central complex (k+p) was 6.6 s-1. The release of the steroid product androsterone (kPr) occurred at a comparable rate of 10.5 s-1. The main contribution to the overall kcat of 0.42 s-1 came from the release of the NADP+ product, kQr = 0.65 s-1.TABLE 2Estimates of rate constants for the individual steps described in Scheme 1Kinetic constantValuekAb (μm-1s-1)aRate constants were calculated with the following equations (28): kAb = kcatRed/KmNADPH; kAr = kcatRedKiNADPH/KmNADPH; kQr = kcatOxiKiNADP+/KmNADP+, and kQb = kcatOxi/KmNADP+5.1kAr (s-1)aRate constants were c
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