Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4
2006; Elsevier BV; Volume: 281; Issue: 14 Linguagem: Inglês
10.1074/jbc.m511375200
ISSN1083-351X
AutoresEmre M. Isin, F. Peter Guengerich,
Tópico(s)Analytical Chemistry and Chromatography
ResumoCytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate- and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steady-state kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and α-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity. Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate- and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steady-state kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and α-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity. Cytochrome P450 (P450) 2The abbreviations used are: P450, cytochrome P450 (also termed "heme thiolate P450" (1Palmer G. Reedijk J. J. Biol. Chem. 1992; 267: 665-677Abstract Full Text PDF PubMed Google Scholar)); ITC, isothermal titration calorimetry. 2The abbreviations used are: P450, cytochrome P450 (also termed "heme thiolate P450" (1Palmer G. Reedijk J. J. Biol. Chem. 1992; 267: 665-677Abstract Full Text PDF PubMed Google Scholar)); ITC, isothermal titration calorimetry. enzymes are found throughout nature, from bacteria to humans. These enzymes generally catalyze mixed function oxidation reactions that have similar chemistry or else utilize parts of the general catalytic mechanism for reductions and rearrangements (2Ortiz de Montellano P.R. De Voss J.J. Cytochrome P450: Structure, Mechanism, and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 183-245Crossref Scopus (203) Google Scholar, 3Guengerich F.P. Chem. Res. Toxicol. 2001; 14: 611-650Crossref PubMed Scopus (1322) Google Scholar). The wide diversity of substrates of these enzymes and the basis of catalytic selectivity is a topic of considerable interest in the context of both basic biochemistry and practical applications (4Poulos T.L. Johnson E.F. Cytochrome P450: Structure Mechanism, and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 87-114Crossref Scopus (110) Google Scholar). P450 3A4 is one of the most widely studied of the 57 human P450s (5Guengerich F.P. Cytochrome P450: Structure Mechanism, and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 377-530Crossref Scopus (274) Google Scholar), mainly due to its role in the metabolism of more than one-half of the drugs on the market as well as various endogenous and exogenous molecules (6Williams J.A. Hyland R. Jones B.C. Smith D.A. Hurst S. Goosen T.C. Peterkin V. Koup J.R. Ball S.E. Drug Metab. Dispos. 2004; 32: 1201-1208Crossref PubMed Scopus (742) Google Scholar, 7Guengerich F.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 1-17Crossref PubMed Scopus (1034) Google Scholar). In addition, P450 3A4 is the major P450 expressed in liver (8Shimada T. Yamazaki H. Mimura M. Inui Y. Guengerich F.P. J. Pharmacol. Exp. Ther. 1994; 270: 414-423PubMed Google Scholar) and in the intestine (9Zhang Q.-Y. Dunbar D. Ostrowska A. Zeisloft S. Yang J. Kaminsky L. Drug Metab. Dispos. 1999; 27: 804-809PubMed Google Scholar). Recently solved P450 3A4 crystal structures (10Yano J.K. Wester M.R. Schoch G.A. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 38091-38094Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar, 11Williams P.A. Cosme J. Vinkovic D.M. Ward A. Angove H.C. Day P.J. Vonrhein C. Tickle I.J. Jhoti H. Science. 2004; 305: 683-686Crossref PubMed Scopus (709) Google Scholar, 12Scott E.E. Halpert J.R. Trends Biochem. Sci. 2005; 30: 5-7Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) demonstrate the presence of a large active site, consistent with the broad range of substrates that P450 3A4 can accommodate (7Guengerich F.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 1-17Crossref PubMed Scopus (1034) Google Scholar, 13Rendic S. Drug Metab. Rev. 2002; 34: 83-448Crossref PubMed Scopus (712) Google Scholar), including (in order of increasing size) acetaminophen (14Patten C.J. Thomas P.E. Guy R.L. Lee M. Gonzalez F.J. Guengerich F.P. Yang C.S. Chem. Res. Toxicol. 1993; 6: 511-518Crossref PubMed Scopus (355) Google Scholar) (Mr 151), testosterone (15Guengerich F.P. Martin M.V. Beaune P.H. Kremers P. Wolff T. Waxman D.J. J. Biol. Chem. 1986; 261: 5051-5060Abstract Full Text PDF PubMed Google Scholar) (Mr 288), bromocriptine (16Peyronneau M.A. Delaforge M. Riviere R. Renaud J.P. Mansuy D. Eur. J. Biochem. 1994; 223: 947-956Crossref PubMed Scopus (55) Google Scholar) (Mr 655), and cyclosporin (17Combalbert J. Fabre I. Fabre G. Dalet I. Derancourt J. Cano J.P. Maurel P. Drug Metab. Dispos. 1989; 17: 197-207PubMed Google Scholar) (Mr 1201). Despite its seemingly flexible substrate selectivity, P450 3A4 displays a high degree of regio- and stereoselectivity in many substrate oxidations (18Krauser J.A. Voehler M. Tseng L.-H. Schefer A.B. Godejohann M. Guengerich F.P. Eur. J. Biochem. 2004; 271: 3962-3969Crossref PubMed Scopus (51) Google Scholar, 19Krauser J.A. Guengerich F.P. J. Biol. Chem. 2005; 280: 19496-19506Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). One of the important features of P450 3A4 is its cooperative behavior, manifested in unusual substrate oxidation kinetics. One of the earlier examples is the stimulation of the 8,9-epoxidation of aflatoxin B1 by α-naphthoflavone (20Kapitulnik J. Poppers P.J. Buening M.K. Fortner J.G. Conney A.H. Clin. Pharmacol. Ther. 1977; 22: 475-485Crossref PubMed Scopus (55) Google Scholar, 21Buening M.K. Fortner J.G. Kappas A. Conney A.H. Biochem. Biophys. Res. Commun. 1978; 82: 348-355Crossref PubMed Scopus (58) Google Scholar, 22Shimada T. Guengerich F.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 462-465Crossref PubMed Scopus (344) Google Scholar), which itself is a P450 3A4 substrate. Following this finding, many examples of cooperativity have been reported not only for P450 3A4 but also for human P450s 2C9 (23Hutzler J.M. Hauer M.J. Tracy T.S. Drug Metab. Dispos. 2001; 29: 1029-1034PubMed Google Scholar), 1A2 (24Venkatakrishnan K. Greenblatt D.J. von Moltke L.L. Schmider J. Harmatz J.S. Shader R.I. J. Clin. Pharmacol. 1998; 38: 112-121Crossref PubMed Scopus (140) Google Scholar, 25Miller G.P. Guengerich F.P. Biochemistry. 2001; 40: 7262-7272Crossref PubMed Scopus (61) Google Scholar), and 2B6 (26Ekins S. VandenBranden M. Ring B.J. Wrighton S.A. Pharmacogenetics. 1997; 7: 165-179Crossref PubMed Scopus (104) Google Scholar), providing support that cooperativity is a common feature of multiple P450s (27Ekins S. Ring B.J. Binkley S.N. Hall S.D. Wrighton S.A. Int. J. Clin. Pharmacol. Ther. 1998; 36: 642-651PubMed Google Scholar) and that the effects are substrate- and reaction-dependent (28Stresser D.M. Blanchard A.P. Turner S.D. Erve J.C.L. Dandeneau A.A. Miller V.P. Crespi C.L. Drug Metab. Dispos. 2000; 28: 1440-1448PubMed Google Scholar). Testosterone, 17β-estradiol, aflatoxin B1, and amitriptyline were shown to display homotropic cooperativity (29Ueng Y.-F. Kuwabara T. Chun Y.-J. Guengerich F.P. Biochemistry. 1997; 36: 370-381Crossref PubMed Scopus (383) Google Scholar) (i.e. increasing substrate concentration stimulates oxidation, resulting in sigmoidal velocity versus substrate concentration curves) (30Kuby S.A. A Study of Enzymes. I. CRC Press, Inc., Boca Raton, FL1991: 300Google Scholar). Various flavonoids have been shown to act as effectors to stimulate the oxidation of some substrates, resulting in heterotropic cooperativity (30Kuby S.A. A Study of Enzymes. I. CRC Press, Inc., Boca Raton, FL1991: 300Google Scholar), and to inhibit the oxidation of others (31Hutzler J.M. Tracy T.S. Drug Metab. Dispos. 2002; 30: 355-362Crossref PubMed Scopus (233) Google Scholar). Thus, the interaction of P450 3A4 with ligands is quite complex and can have a significant effect on the observed in vitro oxidation kinetics. Examples of in vivo cooperativity in animal models have been developed (32Tang W. Stearns R.A. Curr. Drug Metab. 2001; 2: 185-198Crossref PubMed Scopus (139) Google Scholar), and an understanding of the underlying mechanisms of cooperative behavior is important in prediction of drug-drug interactions in practical settings (33Egnell A.C. Houston J.B. Boyer C.S. J. Pharmacol. Exp. Therap. 2005; 312: 926-937Crossref PubMed Scopus (33) Google Scholar). Several groups have proposed models to explain the observed cooperativity (34Houston J.B. Galetin A. Arch. Biochem. Biophys. 2005; 433: 351-360Crossref PubMed Scopus (85) Google Scholar). A rather general consensus is that multiple ligands may interact with P450 3A4 simultaneously, although (i) direct physical evidence is very limited (35Dabrowski M.J. Schrag M.L. Wienkers L.C. Atkins W.M. J. Am. Chem. Soc. 2002; 124: 11866-11867Crossref PubMed Scopus (84) Google Scholar) and (ii) the number of substrate/effector molecules bound and binding sites has not been established. One model, based on steady-state kinetics, has simultaneous occupancy of the P450 3A4 active site with two substrates, which may or may not be identical molecules and which are bound to different domains of the active site (36Shou M. Grogan J. Mancewicz J.A. Krausz K.W. Gonzalez F.J. Gelboin H.V. Korzekwa K.R. Biochemistry. 1994; 33: 6450-6455Crossref PubMed Scopus (362) Google Scholar, 37Shou M. Dai R. Cui D. Korzekwa K.R. Baillie T.A. Rushmore T.H. J. Biol. Chem. 2001; 276: 2256-2262Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 38Shou M.G. Methods Enzymol. 2002; 357: 261-276Crossref PubMed Scopus (21) Google Scholar). Alternatively, a three-site model with a distinct effector binding site has been proposed using a similar steady-state kinetic modeling approach (39Kenworthy K.E. Clarke S.E. Andrews J. Houston J.B. Drug Metab. Dispos. 2001; 29: 1644-1651PubMed Google Scholar). Site-directed mutagenesis studies have led to the proposal of a two-substrate/one-effector domain model in which the effector is located in close proximity of the active site (40Harlow G.R. Halpert J.R. J. Biol. Chem. 1997; 272: 5396-5402Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 41He Y.A. Roussel F. Halpert J.R. Arch. Biochem. Biophys. 2003; 409: 92-101Crossref PubMed Scopus (77) Google Scholar). However, all of these models are based on steady-state turnover kinetic data and do not provide detailed information on the contributions of the individual steps of the catalytic cycle to the observed cooperativity. We have previously focused on the substrate binding step of the P450 catalytic cycle in an attempt to understand better the mechanisms involved in cooperativity (42Hosea N.A. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 5929-5939Crossref PubMed Scopus (225) Google Scholar). Recent work from two other groups has also been directed toward substrate binding to P450 3A4 using spectral and EPR approaches (43Baas B.J. Denisov I.G. Sligar S.G. Arch. Biochem. Biophys. 2004; 430: 218-228Crossref PubMed Scopus (151) Google Scholar, 44Roberts A.G. Campbell A.P. Atkins W.M. Biochemistry. 2005; 44: 1353-1366Crossref PubMed Scopus (66) Google Scholar). In this work, we have expanded our investigations of ligand binding to P450 3A4 (29Ueng Y.-F. Kuwabara T. Chun Y.-J. Guengerich F.P. Biochemistry. 1997; 36: 370-381Crossref PubMed Scopus (383) Google Scholar, 42Hosea N.A. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 5929-5939Crossref PubMed Scopus (225) Google Scholar) using transient state kinetics and equilibrium methods, including quantitative dialysis and ITC. Ligand fluorescence was used as a direct approach to monitor ligand-P450 3A4 interactions, in addition to UV-visible observations of heme perturbations. Our studies on the interactions of various molecules with P450 3A4 lead us to propose a three-step binding model in which the first step does not perturb the heme spectrum. We have also examined the relevance of the individual steps of ligand binding to reduction of ferric to ferrous P450 subsequent event in the catalytic cycle of P450 3A4. These studies demonstrate that binding of ligands to P450 3A4 is a complex multistep process and that investigation of ligand binding interactions solely with heme spin state changes can be misleading. Chemicals—α-Naphthoflavone, midazolam, flavone, and bromocriptine were purchased from Sigma. Testosterone was obtained from Steraloids (Newport, RI). All other reagents and solvents were obtained from general commercial suppliers. All chemicals were used without further purification. Spectroscopy—Absorbance spectra were recorded using either an Aminco DW2a/OLIS or a Cary 14/OLIS spectrophotometer (On-Line Instrument Systems, Bogart, GA). Fluorescence measurements were made using a DM45/OLIS spectrofluorimeter. Stopped-flow experiments were carried out using an OLIS RSM-1000 instrument. A 4 × 20-mm cell was used for absorbance measurements, and a 4 × 4-mm cell was used for fluorescence measurements. Enzymes—Recombinant P450 3A4 with a C-terminal His5 tag (45Gillam E.M.J. Baba T. Kim B.-R. Ohmori S. Guengerich F.P. Arch. Biochem. Biophys. 1993; 305: 123-131Crossref PubMed Scopus (380) Google Scholar) was expressed in Escherichia coli and purified as described previously (42Hosea N.A. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 5929-5939Crossref PubMed Scopus (225) Google Scholar). E. coli recombinant rat NADPH-P450 reductase (46Hanna I.H. Teiber J.F. Kokones K.L. Hollenberg P.F. Arch. Biochem. Biophys. 1998; 350: 324-332Crossref PubMed Scopus (179) Google Scholar) and human liver cytochrome b5 (47Guengerich F.P. Arch. Biochem. Biophys. 2005; 440: 204-211Crossref PubMed Scopus (66) Google Scholar) were prepared as described elsewhere. Protocatechuate dioxygenase was a gift from D. P. Ballou (University of Michigan, Ann Arbor, MI). Reconstitution System—For the reduction kinetics experiments and for some of the testosterone binding experiments, P450 3A4 was reconstituted freshly before the experiment by mixing the components in the following order: P450 3A4 (0.5 or 2 μm), NADPH-P450 reductase (1 or 4 μm), cytochrome b5 (0.5 or 2 μm), sodium cholate (0.5 mm), and phospholipid mixture (40 μg/ml, prepared as described previously (48Yamazaki H. Johnson W.W. Ueng Y.-F. Shimada T. Guengerich F.P. J. Biol. Chem. 1996; 271: 27438-27444Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar)). The components were kept at room temperature for 20 min and mixed periodically by gentle shaking. After 20 min, potassium HEPES buffer (50 mm, pH 7.4), glutathione (3 mm), and MgCl2 (30 mm) were added, and the reconstituted system was kept on ice until use. Spectral Binding Titrations—Binding affinities of ligands to P450 3A4 were determined (at 23 °C) by titrating 1 μm enzyme with the ligand, in a total volume of 1.0 ml of 100 mm potassium phosphate buffer (pH 7.4). Final CH3OH concentrations were ≤2% (v/v). The reference cuvette, containing an equal concentration of enzyme in buffer, was titrated with an equal volume of the vehicle solvent. UV-visible spectra (350–500 nm) were recorded after each addition, and the absorbance differences (at the wavelength maximum and minimum) were plotted against the added ligand concentrations. Spectral dissociation constants (Ks) were estimated using GraphPad Prism software (GraphPad Software, San Diego, CA) or DynaFit (49Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1329) Google Scholar) simulation software (Biokin, Pullman, WA). Unless the estimated Ks was within 5-fold of the P450 concentration, a nonlinear regression analysis was applied using the hyperbolic equation ΔA = Bmax[L]/(Ks + [L]), where A is the absorbance difference, Bmax is the maximum absorbance difference extrapolated to infinite ligand concentration, and [L] is the ligand concentration. For the high affinity ligand bromocriptine, a quadratic equation was used to correct for the bound enzyme concentration: ΔA = A0 + (Bmax/2[E])((Ks + [E] + [L]) – ((Ks + [E] + [L])2 – 4[E][L])1/2), with E being the total enzyme concentration and A0 being a coefficient in each analysis and not relevant. Alternatively, DynaFit simulation software was used with a one-step enzyme-ligand binding model, yielding identical results obtained from the hyperbolic or quadratic nonlinear regression analyses. With flavone, the best fit was obtained with the Hill equation ΔA=Bmax[L]n/(Ksn+[L]n), where n is a measure of cooperativity. Binding Kinetics of Ligands to P450 3A4—In the stopped-flow experiments, either the changes in heme spectra were monitored as a function of time (in the absorbance mode), or the fluorescence quenching of ligands was monitored (in the fluorescence mode). One of the drive syringes contained purified P450 3A4, diluted to 2 or 4 μm in 100 mm potassium phosphate buffer (pH 7.4). The second drive syringe contained the ligand solution (bromocriptine, dissolved in 0.10 m HCl, was diluted in buffer to the desired concentration, and all other ligands were dissolved in CH3OH with ≤2% (v/v) final CH3OH concentration). All stopped-flow measurements were carried out at 23 °C. Immediately after mixing equal amounts (150 μl) of reagent from both syringes, UV-visible spectra (350–500 nm) were collected in the rapid scanning mode with a 16 × 1-mm scan disk, which is a component of the instrument and is used to acquire spectra rapidly. Depending on the data collection time, between 10 and 1000 scans/s were acquired; generally, averages of four experiments were used in the subsequent data analyses. Time-resolved spectra were collected with at least five ligand concentrations for each ligand. Kinetic traces were extracted from the acquired spectra, utilizing either ΔA390 or ΔA390 – A418 (or ΔA390 – A420) and were analyzed using the manufacturer's software (OLIS), GraphPad Prism, or DynaFit. Binding kinetics of testosterone and bromocriptine to P450 3A4 were also measured using the reconstituted system. In these experiments, one of the drive syringes contained P450 3A4 (2 μm), NADPH-P450 reductase (4 μm), cytochrome b5 (2 μm), and other components of the reconstitution system. The second drive syringe contained the testosterone or the bromocriptine solution. In the fluorescence experiments, after mixing the contents of the drive syringes, emission spectra were collected as a function of time with a midplane photomultiplier tube and using the 16 × 1-mm scan disk. The excitation wavelength was 325 nm for bromocriptine and α-naphthoflavone. In general, an excitation monochromator slit of 1.24 mm was used, corresponding to an 8-nm bandpass. Alternatively, data were collected using a >385-nm long pass filter in the single wavelength mode, and the kinetic traces were analyzed using the OLIS software or GraphPad Prism. Generally, averages of four experiments were used in the subsequent data analyses, and S.E. values indicate the goodness of the fit to the average of the multiple experiments. Anaerobic Experiments—The basic set-up (50Burleigh Jr., B.D. Foust G.P. Williams Jr., C.H. Anal. Biochem. 1969; 27: 536-544Crossref PubMed Scopus (63) Google Scholar) and recent modifications (51Guengerich F.P. Krauser J.A. Johnson W.W. Biochemistry. 2004; 43: 10775-10788Crossref PubMed Scopus (78) Google Scholar) have been described elsewhere. Anaerobic conditions were achieved by connecting the glass tonometers to a gas train via a manifold and alternating between vacuum and argon for 10 cycles. In the reduction experiments, an additional three vacuum-CO cycles were carried out prior to loading the drive syringes. An O2 scrubbing system was included in the glass tonometers consisting of protocatechuate dioxygenase (0.7 μm) and protocatechuate (80 μm, added into the solution after 5 vacuum/argon cycles, through a side arm) (52Patil P.V. Ballou D.P. Anal. Biochem. 2000; 286: 187-192Crossref PubMed Scopus (91) Google Scholar). The drive syringes and lines of the stopped-flow apparatus were depleted of O2 using an overnight procedure described in detail previously (53Yun C.-H. Kim K.-H. Calcutt M.W. Guengerich F.P. J. Biol. Chem. 2005; 280: 12279-12291Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Reduction Kinetics—The reduction rate of ferric (Fe3+) to ferrous (Fe2+) P450 was measured at 23 °C with the increase in absorbance at 450 nm or the decrease at 390 nm. Glass tonometers (under a positive CO atmosphere and containing the O2 scrubbing system; see above) were used to fill the drive syringes of the stopped-flow instrument, with minimum exposure to the atmosphere. One of the syringes contained an anaerobic solution of P450 3A4 (0.5 μm, reconstituted as described above), and the second syringe contained 0.4 mm NADPH. In some reduction experiments, bromocriptine (10 μm) was also included either in the syringe containing the enzyme or the one containing NADPH. After mixing the components of the two syringes, UV-visible spectra (355–580 nm) were collected, and kinetic traces were extracted at the wavelengths of interest and analyzed further using GraphPad Prism. As in the case of binding kinetics experiments, between 10 and 1000 scans/s were acquired, depending on the data collection time, and generally averages of four experiments were used in the subsequent data analyses. Equilibrium Dialysis—The stoichiometry of bromocriptine binding to P450 3A4 was examined using five-cavity equilibrium dialysis cells (Bel-Art Products, Pequannock, NJ). A dialysis membrane with a 12–14-kDa cut-off was placed between the two halves of the dialysis cell. The solutions (900 μl) were loaded into the cavities using a 1-ml syringe. Both sides contained an equal amount of bromocriptine solution (0–5 μm in 100 mm potassium phosphate buffer, pH 7.4, diluted from a 300 μm bromocriptine stock solution in 0.10 m HCl). In addition to bromocriptine, one side also contained P450 3A4 (1.0 μm). The solutions were equilibrated for 24 h at room temperature with mechanical rocking. After equilibration, 800-μl aliquots were removed from the cavities, and 100 μl of 25% HClO4 was added. The solutions were mixed using a vortex device and centrifuged (2 × 103× g, 10 min), and 750-μl aliquots were removed. The aliquots were diluted to 2.0 ml with H2O, and fluorescence spectra (350–550 nm) were recorded using mirror-coated fluorimeter cuvettes (Starna Cells, Atascadero, CA). The excitation wavelength was 325 nm, and emission at 420 nm was used to quantify the amount of bromocriptine. ITC—ITC titrations were carried out at 25 °C using a VP-ITC instrument (MicroCal, Northampton, MA). Prior to the titration, P450 3A4 was dialyzed twice against 100 volumes of 100 mm potassium phosphate buffer (pH 7.4) at 4 °C for 4 h. Following the dialysis, the P450 concentration was determined spectrally using the method of Omura and Sato (54Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Abstract Full Text PDF PubMed Google Scholar). The reference cell of the ITC instrument was filled with dialysis buffer. In a typical experiment, the bromocriptine solution (5 μm in dialysis buffer, diluted from 300 μm bromocriptine stock solution in 0.10 m HCl) in the ITC cell was titrated with P450 3A4 (35 μm) loaded into the ITC syringe. The first injection (2 μl, omitted from analysis) was followed by 25 injections of 10 μl, with 8-min intervals between injections. The cell contents were stirred at 450 rpm to provide immediate mixing. The thermal power (heat per unit time) required to keep the cell temperature constant was monitored with time. The peaks observed in the power versus time plots (thermograms, not shown) were integrated using the ORIGIN software (MicroCal). The heats of dilution were obtained from the saturating part of the thermograms and subtracted from each integrated peak. The total heat change was plotted versus the concentration of P450 added to give a titration curve (binding isotherm), expressed in units of kJ mol–1. Kinetic Modeling of Data—Kinetic binding data were fit to various proposed models using DynaFit software (49Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1329) Google Scholar). Rate constants were estimated by globally fitting the kinetic data at six different ligand concentrations to the proposed models. The initial ligand concentrations were allowed to float within 10% of the starting value in the system, and in most cases the concentration adjustment was less than 5%. Sample scripts are included in the supplemental data. Spectral Equilibrium Binding Titrations—Affinities of various ligands with P450 3A4 were first estimated spectrophotometrically by monitoring the heme spectral changes upon the addition of ligands. Ks values were obtained from the titration curves, as described under "Experimental Procedures." Ks values estimated previously in this laboratory and the Ks values determined for the new ligands included in this study are summarized in Table 1.TABLE 1Binding rates and affinities of ligands to P450 3A4LigandConcentration used for initial analysisSoret shiftaFrom Ref. 55.KskabFrom initial analysis. ΔA = Aae-kat + Abe-kbt.kbbFrom initial analysis. ΔA = Aae-kat + Abe-kbt.AabFrom initial analysis. ΔA = Aae-kat + Abe-kbt.AbbFrom initial analysis. ΔA = Aae-kat + Abe-kbt.Apparent second order k rate constantApparent second order k rate constant (ΔF325/>385)μmμms-1m-1 s-1Testosterone100I41 ± 11 (n = 1.3 ± 0.1)cFrom Ref. 42.,dFit to the Hill equation, ΔA=Bmax[L]n/(Ksn+[L]n) with n values given in parentheses.37 ± 13.8 ± 0.10.023 ± 0.0010.014 ± 0.0011.7 ± 0.1 × 105—e—, nonfluorescent.Midazolam75I8.5 ± 0.2cFrom Ref. 42.21 ± 13.3 ± 0.10.015 ± 0.0010.025 ± 0.0011.3 ± 0.3 × 105—Bromocriptine1.5I0.40 ± 0.050.85 ± 0.020.09 ± 0.010.015 ± 0.0010.049 ± 0.0014.3 ± 0.5 × 1054.0 ± 0.5 × 106Flavone100I27.0 ± 0.9 (n = 1.9 ± 0.1)dFit to the Hill equation, ΔA=Bmax[L]n/(Ksn+[L]n) with n values given in parentheses.46 ± 60.76 ± 0.04fRate of reverse Type I shift.0.013 ± 0.0010.006 ± 0.0012.5 ± 1.3 × 105—a From Ref. 55Schenkman J.B. Remmer H. Estabrook R.W. Mol. Pharmacol. 1967; 3: 113-123PubMed Google Scholar.b From initial analysis. ΔA = Aae-kat + Abe-kbt.c From Ref. 42Hosea N.A. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 5929-5939Crossref PubMed Scopus (225) Google Scholar.d Fit to the Hill equation, ΔA=Bmax[L]n/(Ksn+[L]n) with n values given in parentheses.e —, nonfluorescent.f Rate of reverse Type I shift. Open table in a new tab The P450 3A4 substrates testosterone, midazolam, flavone, α-naphthoflavone, and bromocriptine all produced a Type I shift in the heme Soret band, reflecting the displacement of H2O as the sixth ligand and resulting in a low to high spin change in the P450 iron (55Schenkman J.B. Remmer H. Estabrook R.W. Mol. Pharmacol. 1967; 3: 113-123PubMed Google Scholar). Bromocriptine, the most bulky of the substrates studied, had the highest apparent affinity (Ks = 0.4 μm), consistent with a previously reported dissociation constant (16Peyronneau M.A. Delaforge M. Riviere R. Renaud J.P. Mansuy D. Eur. J. Biochem. 1994; 223: 947-
Referência(s)