Cytochrome b 5 Augments the 17,20-Lyase Activity of Human P450c17 without Direct Electron Transfer
1998; Elsevier BV; Volume: 273; Issue: 6 Linguagem: Inglês
10.1074/jbc.273.6.3158
ISSN1083-351X
AutoresRichard J. Auchus, Tim C. Lee, Walter L. Miller,
Tópico(s)Hormonal Regulation and Hypertension
ResumoIn the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17α-hydroxylation of 21-carbon steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19sex steroids. Cytochrome b 5 augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17, human P450-oxidoreductase (OR), and/or human cytochromeb 5 in Saccharomyces cerevisiae and analyzed the 17α-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17α-hydroxylase and trace 17,20-lyase activities toward both Δ4 and Δ5 steroids. Coexpression of human OR with P450c17 increases the V max of both the 17α-hydroxylase and 17,20-lyase reactions 5-fold; coexpression of human b 5 with P450c17 also increases theV max of the 17,20-lyase reactions but not of the 17α-hydroxylase reactions. Simultaneous expression of humanb 5 with P450c17 and OR, or addition of purified human b 5 to microsomes from yeast coexpressing human P450c17 and OR, further increases theV max of the 17,20-lyase reaction without altering 17α-hydroxylase activity. Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b 5, apo-b 5, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action ofb 5 does not require electron transfer fromb 5 to P450c17. These data suggest that humanb 5 acts principally as an allosteric effector that interacts primarily with the P450c17·OR complex to stimulate 17,20-lyase activity. In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17α-hydroxylation of 21-carbon steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19sex steroids. Cytochrome b 5 augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17, human P450-oxidoreductase (OR), and/or human cytochromeb 5 in Saccharomyces cerevisiae and analyzed the 17α-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17α-hydroxylase and trace 17,20-lyase activities toward both Δ4 and Δ5 steroids. Coexpression of human OR with P450c17 increases the V max of both the 17α-hydroxylase and 17,20-lyase reactions 5-fold; coexpression of human b 5 with P450c17 also increases theV max of the 17,20-lyase reactions but not of the 17α-hydroxylase reactions. Simultaneous expression of humanb 5 with P450c17 and OR, or addition of purified human b 5 to microsomes from yeast coexpressing human P450c17 and OR, further increases theV max of the 17,20-lyase reaction without altering 17α-hydroxylase activity. Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b 5, apo-b 5, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action ofb 5 does not require electron transfer fromb 5 to P450c17. These data suggest that humanb 5 acts principally as an allosteric effector that interacts primarily with the P450c17·OR complex to stimulate 17,20-lyase activity. Among the many chemical transformations catalyzed by cytochrome P450 enzymes, steroid hormone hydroxylations, and cleavages are of particular interest because of their mechanistic complexities and essential roles in physiology (1Miller W.L. Endocr. Rev. 1988; 9: 295-318Crossref PubMed Scopus (1170) Google Scholar). P450c17 catalyzes both 17α-hydroxylase and 17,20-lyase activities (2Nakajin S. Hall P.F. J. Biol. Chem. 1981; 256: 3871-3876Abstract Full Text PDF PubMed Google Scholar) (for review see Ref.3Miller W.L. Auchus R.J. Geller D.H. Steroids. 1997; 62: 135-144Crossref Scopus (203) Google Scholar) and also has a modest degree of 16α-hydroxylase activity (4Nakajin S. Takahashi M. Shinoda M. Hall P.F. Biochem. Biophys. Res. Commun. 1985; 132: 708-713Crossref PubMed Scopus (69) Google Scholar). In human beings, the 17α-hydroxylase reaction leads to the glucocorticoid, cortisol, and the subsequent 17,20-lyase reaction leads to precursors of sex steroids. As the sole pathway leading to biosynthesis of circulating sex steroids, the regulation of this 17,20-lyase activity is central to understanding the developmental regulation of dehydroepiandrosterone sulfate (DHEA) 1The abbreviations used are: DHEA, dehydroepiandrosterone; OR, P450-oxidoreductase;b 5, cytochrome b 5; V10, vector pYeDP10; V60, vector pYeDP60; cDE2, vector pYcDE-2; PCR, polymerase chain reaction. 1The abbreviations used are: DHEA, dehydroepiandrosterone; OR, P450-oxidoreductase;b 5, cytochrome b 5; V10, vector pYeDP10; V60, vector pYeDP60; cDE2, vector pYcDE-2; PCR, polymerase chain reaction. with adrenarche and aging, and to the pathogenesis of the polycystic ovary syndrome (3Miller W.L. Auchus R.J. Geller D.H. Steroids. 1997; 62: 135-144Crossref Scopus (203) Google Scholar). The 17,20-lyase activity, involving the oxidative cleavage of a carbon-carbon bond, is regulated in a tissue-specific and developmentally programmed manner by factors such as the abundance of the electron donor flavoprotein P450-oxidoreductase (OR) (5Lin D. Black S.M. Nagahama Y. Miller W.L. Endocrinology. 1993; 132: 2498-2506Crossref PubMed Google Scholar, 6Yanagibashi K. Hall P.F. J. Biol. Chem. 1986; 261: 8429-8433Abstract Full Text PDF PubMed Google Scholar), the co-existence of 3β-hydroxysteroid dehydrogenase and P450c21 (7Conley A.J. Bird I.M. Biol. Reprod. 1997; 56: 789-799Crossref PubMed Scopus (249) Google Scholar), and post-translational modification of P450c17 (8Zhang L. Rodriguez H. Ohno S. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10619-10623Crossref PubMed Scopus (398) Google Scholar).To perform catalysis, P450c17, like all other microsomal P450 oxygenases, must receive two electrons from NADPH via OR. Cytochromeb 5 has also been implicated as a component of the 17,20-lyase reaction, as b 5 augments 17,20-lyase activity and occasionally 17α-hydroxylase activity of P450c17 in reconstituted systems (9Onoda M. Hall P.F. Biochem. Biophys. Res. Commun. 1982; 108: 454-460Crossref PubMed Scopus (117) Google Scholar, 10Kominami S. Ogawa N. Morimune R. Huang D.Y. Takemori S. J. Steroid Biochem. Mol. Biol. 1992; 42: 57-64Crossref PubMed Scopus (84) Google Scholar); however, our laboratory could not confirm this effect in transfected monkey kidney COS-1 cells (5Lin D. Black S.M. Nagahama Y. Miller W.L. Endocrinology. 1993; 132: 2498-2506Crossref PubMed Google Scholar). Inconsistencies in the animal species of P450c17, OR, and b 5 used in previous studies preclude extrapolation of the available biochemical data to human adrenal and gonadal physiology; furthermore, the mechanism(s) of these reportedb 5-mediated increases in 17,20-lyase activity remain unknown.Among the various systems developed to study mammalian cytochromes P450, transfection of genetically modified yeast cells provides the opportunity to study the activities of a cytochrome P450 in the presence of various combinations of electron transfer proteins in the native microsomal environment (11Pompon D. Louerat B. Bronine A. Urban P. Methods Enzymol. 1996; 272: 51-64Crossref PubMed Google Scholar). To clarify the function of cytochrome b 5 in 17,20-lyase activity, we systematically varied the abundance of putative electron transfer proteins in yeast microsomes containing human P450c17. We find that human, but not yeast cytochrome b 5 can selectively augment the rate of the 17,20-lyase reaction by more than 10-fold. However, this augmentation requires OR and occurs without electron transfer to or from cytochrome b 5.DISCUSSIONThe 17,20-lyase/17α-hydroxylase ratio in the human adrenal rises dramatically with the onset of adrenarche at age 8–10, reaches maximal values at age 25–35, and then falls progressively with aging (39Orentreich N. Brind J.L. Rizer R.L. Vogelman J.H. J. Clin. Endocrinol. Metab. 1984; 59: 551-555Crossref PubMed Scopus (1258) Google Scholar); as these phenomena occur only in human beings and great apes (40Cutler G.B. Glenn M. Bush M. Hodgen G.D. Graham C.E. Loriaux D.L. Endocrinology. 1978; 103: 2112-2118Crossref PubMed Scopus (269) Google Scholar), their study is difficult. These selective, physiologic, developmentally programmed changes in human adrenal 17,20-lyase activity imply regulatory mechanisms beyond transcription of P450c17 or OR (3Miller W.L. Auchus R.J. Geller D.H. Steroids. 1997; 62: 135-144Crossref Scopus (203) Google Scholar, 8Zhang L. Rodriguez H. Ohno S. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10619-10623Crossref PubMed Scopus (398) Google Scholar). Most P450 enzymes catalyze multiple reactions, but the ratio of their activities remains fixed. The developmentally and possibly hormonally programmed changes in the ratio of 17,20-lyase to 17α-hydroxylase activities of human P450c17 provide a unique system for studying the differential regulation of two reactions catalyzed by a single P450 enzyme.An augmentation of the 17,20-lyase activity of P450c17 byb 5 has been observed in vitro (9Onoda M. Hall P.F. Biochem. Biophys. Res. Commun. 1982; 108: 454-460Crossref PubMed Scopus (117) Google Scholar, 25Katagiri M. Kagawa N. Waterman M.R. Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (204) Google Scholar) but was not seen in transfected COS-1 cells (5Lin D. Black S.M. Nagahama Y. Miller W.L. Endocrinology. 1993; 132: 2498-2506Crossref PubMed Google Scholar), possibly because the endogenous b 5 in those cells was sufficient to stimulate 17,20-lyase activity maximally. Thus, it has not been clear how or if b 5 regulates human P450c17 activitiesin vivo. The use of microsomes from yeast engineered to express human P450c17, OR, or b 5 from inducible promoters permits the quantitative manipulation of each protein in a membrane environment that should simulate events in vivo. This permits greater experimental flexibility than the use of bicistronic plasmids (41Parikh A. Gillam E.M.J. Guengerich F.P. Nature Biotech. 1997; 15: 346-354Crossref Scopus (279) Google Scholar), fusion proteins (29Shet M.S. Fisher C.W. Arlotto M.P. Shackleton C.H.L. Holmans P.L. Martin-Wixtrom C.A. Saeki Y. Estabrook R.W. Arch. Biochem. Biophys. 1994; 311: 402-417Crossref PubMed Scopus (68) Google Scholar), or viral vectors (42Aoyama T. Nagata K. Yamazoe Y. Kato R. Matsunaga E. Gelboin H.V. Gonzalez F.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5425-5429Crossref PubMed Scopus (63) Google Scholar), and obviates concerns about the relevance of data from detergent-solubilized systems to in vivo systems.Titration experiments with purified human holo-b 5, apo-b 5, and cytochrome c showed that the stimulatory effect ofb 5 on 17,20-lyase activity is not mediated by electron transfer from b 5 and suggest thatb 5 exerts an allosteric effect on the P450c17·OR complex. This proposed mechanism could explain three observations from other laboratories. First, b 5facilitates electron transfer from OR to P450 3A4 only when all three proteins are premixed before adding NADPH and substrate, but not whenb 5 is premixed with P450 3A4 and added to OR, NADPH, and substrate in stop-flow experiments (24Yamazaki 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 (187) Google Scholar). These data suggested that the stimulatory action of b 5 on testosterone 6β-hydroxylation by P450 3A4 was an allosteric effect and was not mediated by an action of b 5 as an alternate electron donor (24Yamazaki 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 (187) Google Scholar). Second, b 5 is a more potent stimulator of 17,20-lyase activity when the abundance of OR is low, and this stimulation is quite sensitive to small changes in these low amounts of OR (10Kominami S. Ogawa N. Morimune R. Huang D.Y. Takemori S. J. Steroid Biochem. Mol. Biol. 1992; 42: 57-64Crossref PubMed Scopus (84) Google Scholar). Our results corroborate these studies and suggest that b 5 interacts primarily with the P450c17·OR complex and not with P450c17 alone. Third, the redox-active core 1 segment of porcine b 5 alone cannot augment the 17,20-lyase activity of human P450c17 (43Lee-Robichaud P. Kaderbhai M.A. Kaderbhai N. Wright J.N. Akhtar M. Biochem. J. 1997; 321: 857-863Crossref PubMed Scopus (44) Google Scholar), consistent with our findings that electron transfer from humanb 5 is not required to stimulate 17,20-lyase activity.Three conclusions about human physiology emerge from our analysis of the kinetics of human P450c17. First, human androgen biosynthesis proceeds predominantly through the pathway 17α-hydroxypregnenolone → DHEA → androstenedione, rather than through the pathway 17α-hydroxypregnenolone → 17α-hydroxyprogesterone → androstenedione. The pathway via DHEA predominates because the apparentK m for Δ4 17α-hydroxyprogesterone is about 10-fold higher and its V max is one-tenth as fast as the corresponding values for Δ517α-hydroxypregnenolone. Thus, the catalytic efficiencyV max/K m for the 17,20-lyase reaction is nearly 100-fold greater for Δ517α-hydroxypregnenolone than for Δ417α-hydroxyprogesterone. Second, significant androgen biosynthesis via the Δ4 pathway can only occur in the presence of very high Δ4 17α-hydroxyprogesterone concentrations, as found in untreated patients with 21-hydroxylase deficiency (44Miller W.L. Morel Y. Annu. Rev. Genet. 1989; 23: 371-393Crossref PubMed Scopus (110) Google Scholar). Third, considerable microsomal 17,20-lyase activity is found even in the complete absence of b 5; therefore, b 5 deficiency cannot cause a syndrome of complete 17,20-lyase deficiency (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar) as has been suggested (45Giordano S.J. Kaftory A. Steggles A.W. Hum. Genet. 1994; 93: 568-570Crossref PubMed Scopus (59) Google Scholar).The structural nature of the interaction of P450c17 with OR is not known, but the x-ray crystal structures of rat OR (46Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar) and P450-BMP (47Ravichandran K.G. Boddupalli S.S. Hasemann C.A. Peterson J.A. Deisenhofer J. Science. 1993; 261: 731-736Crossref PubMed Scopus (904) Google Scholar) provide useful clues. The redox-partner binding site for P450-BMP, a Type II (microsomal) P450, comprises the surface surrounding a depression in the “proximal” face of the protein that extends down to the face of the heme opposite the substrate-binding pocket (47Ravichandran K.G. Boddupalli S.S. Hasemann C.A. Peterson J.A. Deisenhofer J. Science. 1993; 261: 731-736Crossref PubMed Scopus (904) Google Scholar, 48Hasemann C.A. Kurumbail R.G. Boddupalli S.S. Peterson J.A. Deisenhofer J. Structure. 1995; 3: 41-62Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). This crevasse is lined on one side with positively charged residues from the J′ and K helices (in P450-BMP, lysines 325, 328, and 331) which appear to participate in electrostatic pairing with negatively charged residues in OR. Molecular modeling shows that human P450c17 has a similarly located crevasse of positively charged residues that interact with redox partners (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar). The electron-donating FMN moiety of rat OR also lies at the base of a concave cleft formed by the butterfly-shaped apposition of the FMN and FAD domains (46Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar). However, the FMN domain joins the remainder of the protein via a disordered, flexible hinge that must flex about 90° for the FMN moiety to extend out from the concave cleft of OR (46Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar) to approach the concave redox-partner binding site of P450c17 (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar).Because b 5 normally participates in redox reactions such as methemoglobin reduction (49Kuma F. Prough R.A. Masters B.S. Arch. Biochem. Biophys. 1976; 172: 600-607Crossref PubMed Scopus (45) Google Scholar) and stearyl-CoA desaturation (50Oshino N. Imai Y. Sato R. J. Biochem. (Tokyo). 1971; 69: 155-167Crossref PubMed Scopus (313) Google Scholar) and can serve as an alternate electron donor in some other P450 reactions (51Yamazaki H. Gillam E.M. Dong M.S. Johnson W.W. Guengerich F.P. Shimada T. Arch. Biochem. Biophys. 1997; 342: 329-337Crossref PubMed Scopus (129) Google Scholar), our demonstration thatb 5 serves a role as an allosteric facilitator of electron transfer from OR to P450c17 was unexpected. The binding of redox partners to P450c17 must transmit subtle changes to the substrate-binding pocket, as evidenced by the lowerK m values for Δ5 substrates in the presence of human OR (Table III) and by analogy to the altered regiospecificity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine oxygenation by P450 2D6 in the presence of OR (28Modi S. Gilham D.E. Sutcliffe M.J. Lian L.Y. Primrose W.U. Wolf C.R. Roberts G.C. Biochemistry. 1997; 36: 4461-4470Crossref PubMed Scopus (88) Google Scholar). Furthermore, the oxidative scission of the C17-C20 bond of 17α-hydroxypregnenolone appears to impose much more stringent constraints on the active-site topology of P450c17 than does the 17α-hydroxylase reaction. Therefore, we propose thatb 5 optimizes the geometry of the P450c17·OR complex for the more sensitive 17,20-lyase reaction perhaps by forming a ternary complex (Fig. 7). The structural core 2 domain of b 5 may be the region that stimulates 17,20-lyase activity, as core 2 adopts a similar conformation in the NMR structures of both isolated holo-b 5 (52Muskett F.W. Kelly G.P. Whitford D. J. Mol. Biol. 1996; 258: 172-189Crossref PubMed Scopus (47) Google Scholar) and apo-b 5(53Falzone C.J. Mayer M.R. Whiteman E.L. Moore C.D. Lecomte J.T. Biochemistry. 1996; 35: 6519-6526Crossref PubMed Scopus (81) Google Scholar), whereas the heme-binding core 1 domain is disordered in apo-b 5 (53Falzone C.J. Mayer M.R. Whiteman E.L. Moore C.D. Lecomte J.T. Biochemistry. 1996; 35: 6519-6526Crossref PubMed Scopus (81) Google Scholar). Furthermore, core 2 retains its overall topology during molecular dynamics simulations of apo-b 5, while core 1 loses secondary structure and exhibits conformational mobility (54Storch E.M. Daggett V. Biochemistry. 1996; 35: 11596-11604Crossref PubMed Scopus (31) Google Scholar).A role for b 5 as an allosteric effector protein is consistent with our observation that serine phosphorylation of P450c17 selectively increases 17,20-lyase activity (8Zhang L. Rodriguez H. Ohno S. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10619-10623Crossref PubMed Scopus (398) Google Scholar) and that mutations of arginine residues in the redox-partner binding site of human P450c17 cause isolated 17,20-lyase deficiency (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar). The precise orientation of OR in the electron-donor docking region of P450c17 required to assemble the active oxygenating complex for the 17,20-lyase reaction is impaired by mutation of this surface and enhanced byb 5 or apo-b 5. Phosphorylation of P450c17 probably favors assembly of productive complexes so that electron transfer is more rapid and coupling efficiency is higher; however, the exact mechanism by which phosphorylated serine residues enhance 17,20-lyase activity is not yet known. A more detailed understanding of these complexes is essential for understanding the regulation of 17,20-lyase activity; this in turn may permit development of agents to inhibit this activity, which will aid in the treatment of sex steroid-dependent malignancies and disorders of androgen excess. Among the many chemical transformations catalyzed by cytochrome P450 enzymes, steroid hormone hydroxylations, and cleavages are of particular interest because of their mechanistic complexities and essential roles in physiology (1Miller W.L. Endocr. Rev. 1988; 9: 295-318Crossref PubMed Scopus (1170) Google Scholar). P450c17 catalyzes both 17α-hydroxylase and 17,20-lyase activities (2Nakajin S. Hall P.F. J. Biol. Chem. 1981; 256: 3871-3876Abstract Full Text PDF PubMed Google Scholar) (for review see Ref.3Miller W.L. Auchus R.J. Geller D.H. Steroids. 1997; 62: 135-144Crossref Scopus (203) Google Scholar) and also has a modest degree of 16α-hydroxylase activity (4Nakajin S. Takahashi M. Shinoda M. Hall P.F. Biochem. Biophys. Res. Commun. 1985; 132: 708-713Crossref PubMed Scopus (69) Google Scholar). In human beings, the 17α-hydroxylase reaction leads to the glucocorticoid, cortisol, and the subsequent 17,20-lyase reaction leads to precursors of sex steroids. As the sole pathway leading to biosynthesis of circulating sex steroids, the regulation of this 17,20-lyase activity is central to understanding the developmental regulation of dehydroepiandrosterone sulfate (DHEA) 1The abbreviations used are: DHEA, dehydroepiandrosterone; OR, P450-oxidoreductase;b 5, cytochrome b 5; V10, vector pYeDP10; V60, vector pYeDP60; cDE2, vector pYcDE-2; PCR, polymerase chain reaction. 1The abbreviations used are: DHEA, dehydroepiandrosterone; OR, P450-oxidoreductase;b 5, cytochrome b 5; V10, vector pYeDP10; V60, vector pYeDP60; cDE2, vector pYcDE-2; PCR, polymerase chain reaction. with adrenarche and aging, and to the pathogenesis of the polycystic ovary syndrome (3Miller W.L. Auchus R.J. Geller D.H. Steroids. 1997; 62: 135-144Crossref Scopus (203) Google Scholar). The 17,20-lyase activity, involving the oxidative cleavage of a carbon-carbon bond, is regulated in a tissue-specific and developmentally programmed manner by factors such as the abundance of the electron donor flavoprotein P450-oxidoreductase (OR) (5Lin D. Black S.M. Nagahama Y. Miller W.L. Endocrinology. 1993; 132: 2498-2506Crossref PubMed Google Scholar, 6Yanagibashi K. Hall P.F. J. Biol. Chem. 1986; 261: 8429-8433Abstract Full Text PDF PubMed Google Scholar), the co-existence of 3β-hydroxysteroid dehydrogenase and P450c21 (7Conley A.J. Bird I.M. Biol. Reprod. 1997; 56: 789-799Crossref PubMed Scopus (249) Google Scholar), and post-translational modification of P450c17 (8Zhang L. Rodriguez H. Ohno S. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10619-10623Crossref PubMed Scopus (398) Google Scholar). To perform catalysis, P450c17, like all other microsomal P450 oxygenases, must receive two electrons from NADPH via OR. Cytochromeb 5 has also been implicated as a component of the 17,20-lyase reaction, as b 5 augments 17,20-lyase activity and occasionally 17α-hydroxylase activity of P450c17 in reconstituted systems (9Onoda M. Hall P.F. Biochem. Biophys. Res. Commun. 1982; 108: 454-460Crossref PubMed Scopus (117) Google Scholar, 10Kominami S. Ogawa N. Morimune R. Huang D.Y. Takemori S. J. Steroid Biochem. Mol. Biol. 1992; 42: 57-64Crossref PubMed Scopus (84) Google Scholar); however, our laboratory could not confirm this effect in transfected monkey kidney COS-1 cells (5Lin D. Black S.M. Nagahama Y. Miller W.L. Endocrinology. 1993; 132: 2498-2506Crossref PubMed Google Scholar). Inconsistencies in the animal species of P450c17, OR, and b 5 used in previous studies preclude extrapolation of the available biochemical data to human adrenal and gonadal physiology; furthermore, the mechanism(s) of these reportedb 5-mediated increases in 17,20-lyase activity remain unknown. Among the various systems developed to study mammalian cytochromes P450, transfection of genetically modified yeast cells provides the opportunity to study the activities of a cytochrome P450 in the presence of various combinations of electron transfer proteins in the native microsomal environment (11Pompon D. Louerat B. Bronine A. Urban P. Methods Enzymol. 1996; 272: 51-64Crossref PubMed Google Scholar). To clarify the function of cytochrome b 5 in 17,20-lyase activity, we systematically varied the abundance of putative electron transfer proteins in yeast microsomes containing human P450c17. We find that human, but not yeast cytochrome b 5 can selectively augment the rate of the 17,20-lyase reaction by more than 10-fold. However, this augmentation requires OR and occurs without electron transfer to or from cytochrome b 5. DISCUSSIONThe 17,20-lyase/17α-hydroxylase ratio in the human adrenal rises dramatically with the onset of adrenarche at age 8–10, reaches maximal values at age 25–35, and then falls progressively with aging (39Orentreich N. Brind J.L. Rizer R.L. Vogelman J.H. J. Clin. Endocrinol. Metab. 1984; 59: 551-555Crossref PubMed Scopus (1258) Google Scholar); as these phenomena occur only in human beings and great apes (40Cutler G.B. Glenn M. Bush M. Hodgen G.D. Graham C.E. Loriaux D.L. Endocrinology. 1978; 103: 2112-2118Crossref PubMed Scopus (269) Google Scholar), their study is difficult. These selective, physiologic, developmentally programmed changes in human adrenal 17,20-lyase activity imply regulatory mechanisms beyond transcription of P450c17 or OR (3Miller W.L. Auchus R.J. Geller D.H. Steroids. 1997; 62: 135-144Crossref Scopus (203) Google Scholar, 8Zhang L. Rodriguez H. Ohno S. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10619-10623Crossref PubMed Scopus (398) Google Scholar). Most P450 enzymes catalyze multiple reactions, but the ratio of their activities remains fixed. The developmentally and possibly hormonally programmed changes in the ratio of 17,20-lyase to 17α-hydroxylase activities of human P450c17 provide a unique system for studying the differential regulation of two reactions catalyzed by a single P450 enzyme.An augmentation of the 17,20-lyase activity of P450c17 byb 5 has been observed in vitro (9Onoda M. Hall P.F. Biochem. Biophys. Res. Commun. 1982; 108: 454-460Crossref PubMed Scopus (117) Google Scholar, 25Katagiri M. Kagawa N. Waterman M.R. Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (204) Google Scholar) but was not seen in transfected COS-1 cells (5Lin D. Black S.M. Nagahama Y. Miller W.L. Endocrinology. 1993; 132: 2498-2506Crossref PubMed Google Scholar), possibly because the endogenous b 5 in those cells was sufficient to stimulate 17,20-lyase activity maximally. Thus, it has not been clear how or if b 5 regulates human P450c17 activitiesin vivo. The use of microsomes from yeast engineered to express human P450c17, OR, or b 5 from inducible promoters permits the quantitative manipulation of each protein in a membrane environment that should simulate events in vivo. This permits greater experimental flexibility than the use of bicistronic plasmids (41Parikh A. Gillam E.M.J. Guengerich F.P. Nature Biotech. 1997; 15: 346-354Crossref Scopus (279) Google Scholar), fusion proteins (29Shet M.S. Fisher C.W. Arlotto M.P. Shackleton C.H.L. Holmans P.L. Martin-Wixtrom C.A. Saeki Y. Estabrook R.W. Arch. Biochem. Biophys. 1994; 311: 402-417Crossref PubMed Scopus (68) Google Scholar), or viral vectors (42Aoyama T. Nagata K. Yamazoe Y. Kato R. Matsunaga E. Gelboin H.V. Gonzalez F.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5425-5429Crossref PubMed Scopus (63) Google Scholar), and obviates concerns about the relevance of data from detergent-solubilized systems to in vivo systems.Titration experiments with purified human holo-b 5, apo-b 5, and cytochrome c showed that the stimulatory effect ofb 5 on 17,20-lyase activity is not mediated by electron transfer from b 5 and suggest thatb 5 exerts an allosteric effect on the P450c17·OR complex. This proposed mechanism could explain three observations from other laboratories. First, b 5facilitates electron transfer from OR to P450 3A4 only when all three proteins are premixed before adding NADPH and substrate, but not whenb 5 is premixed with P450 3A4 and added to OR, NADPH, and substrate in stop-flow experiments (24Yamazaki 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 (187) Google Scholar). These data suggested that the stimulatory action of b 5 on testosterone 6β-hydroxylation by P450 3A4 was an allosteric effect and was not mediated by an action of b 5 as an alternate electron donor (24Yamazaki 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 (187) Google Scholar). Second, b 5 is a more potent stimulator of 17,20-lyase activity when the abundance of OR is low, and this stimulation is quite sensitive to small changes in these low amounts of OR (10Kominami S. Ogawa N. Morimune R. Huang D.Y. Takemori S. J. Steroid Biochem. Mol. Biol. 1992; 42: 57-64Crossref PubMed Scopus (84) Google Scholar). Our results corroborate these studies and suggest that b 5 interacts primarily with the P450c17·OR complex and not with P450c17 alone. Third, the redox-active core 1 segment of porcine b 5 alone cannot augment the 17,20-lyase activity of human P450c17 (43Lee-Robichaud P. Kaderbhai M.A. Kaderbhai N. Wright J.N. Akhtar M. Biochem. J. 1997; 321: 857-863Crossref PubMed Scopus (44) Google Scholar), consistent with our findings that electron transfer from humanb 5 is not required to stimulate 17,20-lyase activity.Three conclusions about human physiology emerge from our analysis of the kinetics of human P450c17. First, human androgen biosynthesis proceeds predominantly through the pathway 17α-hydroxypregnenolone → DHEA → androstenedione, rather than through the pathway 17α-hydroxypregnenolone → 17α-hydroxyprogesterone → androstenedione. The pathway via DHEA predominates because the apparentK m for Δ4 17α-hydroxyprogesterone is about 10-fold higher and its V max is one-tenth as fast as the corresponding values for Δ517α-hydroxypregnenolone. Thus, the catalytic efficiencyV max/K m for the 17,20-lyase reaction is nearly 100-fold greater for Δ517α-hydroxypregnenolone than for Δ417α-hydroxyprogesterone. Second, significant androgen biosynthesis via the Δ4 pathway can only occur in the presence of very high Δ4 17α-hydroxyprogesterone concentrations, as found in untreated patients with 21-hydroxylase deficiency (44Miller W.L. Morel Y. Annu. Rev. Genet. 1989; 23: 371-393Crossref PubMed Scopus (110) Google Scholar). Third, considerable microsomal 17,20-lyase activity is found even in the complete absence of b 5; therefore, b 5 deficiency cannot cause a syndrome of complete 17,20-lyase deficiency (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar) as has been suggested (45Giordano S.J. Kaftory A. Steggles A.W. Hum. Genet. 1994; 93: 568-570Crossref PubMed Scopus (59) Google Scholar).The structural nature of the interaction of P450c17 with OR is not known, but the x-ray crystal structures of rat OR (46Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar) and P450-BMP (47Ravichandran K.G. Boddupalli S.S. Hasemann C.A. Peterson J.A. Deisenhofer J. Science. 1993; 261: 731-736Crossref PubMed Scopus (904) Google Scholar) provide useful clues. The redox-partner binding site for P450-BMP, a Type II (microsomal) P450, comprises the surface surrounding a depression in the “proximal” face of the protein that extends down to the face of the heme opposite the substrate-binding pocket (47Ravichandran K.G. Boddupalli S.S. Hasemann C.A. Peterson J.A. Deisenhofer J. Science. 1993; 261: 731-736Crossref PubMed Scopus (904) Google Scholar, 48Hasemann C.A. Kurumbail R.G. Boddupalli S.S. Peterson J.A. Deisenhofer J. Structure. 1995; 3: 41-62Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). This crevasse is lined on one side with positively charged residues from the J′ and K helices (in P450-BMP, lysines 325, 328, and 331) which appear to participate in electrostatic pairing with negatively charged residues in OR. Molecular modeling shows that human P450c17 has a similarly located crevasse of positively charged residues that interact with redox partners (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar). The electron-donating FMN moiety of rat OR also lies at the base of a concave cleft formed by the butterfly-shaped apposition of the FMN and FAD domains (46Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar). However, the FMN domain joins the remainder of the protein via a disordered, flexible hinge that must flex about 90° for the FMN moiety to extend out from the concave cleft of OR (46Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar) to approach the concave redox-partner binding site of P450c17 (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar).Because b 5 normally participates in redox reactions such as methemoglobin reduction (49Kuma F. Prough R.A. Masters B.S. Arch. Biochem. Biophys. 1976; 172: 600-607Crossref PubMed Scopus (45) Google Scholar) and stearyl-CoA desaturation (50Oshino N. Imai Y. Sato R. J. Biochem. (Tokyo). 1971; 69: 155-167Crossref PubMed Scopus (313) Google Scholar) and can serve as an alternate electron donor in some other P450 reactions (51Yamazaki H. Gillam E.M. Dong M.S. Johnson W.W. Guengerich F.P. Shimada T. Arch. Biochem. Biophys. 1997; 342: 329-337Crossref PubMed Scopus (129) Google Scholar), our demonstration thatb 5 serves a role as an allosteric facilitator of electron transfer from OR to P450c17 was unexpected. The binding of redox partners to P450c17 must transmit subtle changes to the substrate-binding pocket, as evidenced by the lowerK m values for Δ5 substrates in the presence of human OR (Table III) and by analogy to the altered regiospecificity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine oxygenation by P450 2D6 in the presence of OR (28Modi S. Gilham D.E. Sutcliffe M.J. Lian L.Y. Primrose W.U. Wolf C.R. Roberts G.C. Biochemistry. 1997; 36: 4461-4470Crossref PubMed Scopus (88) Google Scholar). Furthermore, the oxidative scission of the C17-C20 bond of 17α-hydroxypregnenolone appears to impose much more stringent constraints on the active-site topology of P450c17 than does the 17α-hydroxylase reaction. Therefore, we propose thatb 5 optimizes the geometry of the P450c17·OR complex for the more sensitive 17,20-lyase reaction perhaps by forming a ternary complex (Fig. 7). The structural core 2 domain of b 5 may be the region that stimulates 17,20-lyase activity, as core 2 adopts a similar conformation in the NMR structures of both isolated holo-b 5 (52Muskett F.W. Kelly G.P. Whitford D. J. Mol. Biol. 1996; 258: 172-189Crossref PubMed Scopus (47) Google Scholar) and apo-b 5(53Falzone C.J. Mayer M.R. Whiteman E.L. Moore C.D. Lecomte J.T. Biochemistry. 1996; 35: 6519-6526Crossref PubMed Scopus (81) Google Scholar), whereas the heme-binding core 1 domain is disordered in apo-b 5 (53Falzone C.J. Mayer M.R. Whiteman E.L. Moore C.D. Lecomte J.T. Biochemistry. 1996; 35: 6519-6526Crossref PubMed Scopus (81) Google Scholar). Furthermore, core 2 retains its overall topology during molecular dynamics simulations of apo-b 5, while core 1 loses secondary structure and exhibits conformational mobility (54Storch E.M. Daggett V. Biochemistry. 1996; 35: 11596-11604Crossref PubMed Scopus (31) Google Scholar).A role for b 5 as an allosteric effector protein is consistent with our observation that serine phosphorylation of P450c17 selectively increases 17,20-lyase activity (8Zhang L. Rodriguez H. Ohno S. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10619-10623Crossref PubMed Scopus (398) Google Scholar) and that mutations of arginine residues in the redox-partner binding site of human P450c17 cause isolated 17,20-lyase deficiency (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar). The precise orientation of OR in the electron-donor docking region of P450c17 required to assemble the active oxygenating complex for the 17,20-lyase reaction is impaired by mutation of this surface and enhanced byb 5 or apo-b 5. Phosphorylation of P450c17 probably favors assembly of productive complexes so that electron transfer is more rapid and coupling efficiency is higher; however, the exact mechanism by which phosphorylated serine residues enhance 17,20-lyase activity is not yet known. A more detailed understanding of these complexes is essential for understanding the regulation of 17,20-lyase activity; this in turn may permit development of agents to inhibit this activity, which will aid in the treatment of sex steroid-dependent malignancies and disorders of androgen excess. The 17,20-lyase/17α-hydroxylase ratio in the human adrenal rises dramatically with the onset of adrenarche at age 8–10, reaches maximal values at age 25–35, and then falls progressively with aging (39Orentreich N. Brind J.L. Rizer R.L. Vogelman J.H. J. Clin. Endocrinol. Metab. 1984; 59: 551-555Crossref PubMed Scopus (1258) Google Scholar); as these phenomena occur only in human beings and great apes (40Cutler G.B. Glenn M. Bush M. Hodgen G.D. Graham C.E. Loriaux D.L. Endocrinology. 1978; 103: 2112-2118Crossref PubMed Scopus (269) Google Scholar), their study is difficult. These selective, physiologic, developmentally programmed changes in human adrenal 17,20-lyase activity imply regulatory mechanisms beyond transcription of P450c17 or OR (3Miller W.L. Auchus R.J. Geller D.H. Steroids. 1997; 62: 135-144Crossref Scopus (203) Google Scholar, 8Zhang L. Rodriguez H. Ohno S. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10619-10623Crossref PubMed Scopus (398) Google Scholar). Most P450 enzymes catalyze multiple reactions, but the ratio of their activities remains fixed. The developmentally and possibly hormonally programmed changes in the ratio of 17,20-lyase to 17α-hydroxylase activities of human P450c17 provide a unique system for studying the differential regulation of two reactions catalyzed by a single P450 enzyme. An augmentation of the 17,20-lyase activity of P450c17 byb 5 has been observed in vitro (9Onoda M. Hall P.F. Biochem. Biophys. Res. Commun. 1982; 108: 454-460Crossref PubMed Scopus (117) Google Scholar, 25Katagiri M. Kagawa N. Waterman M.R. Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (204) Google Scholar) but was not seen in transfected COS-1 cells (5Lin D. Black S.M. Nagahama Y. Miller W.L. Endocrinology. 1993; 132: 2498-2506Crossref PubMed Google Scholar), possibly because the endogenous b 5 in those cells was sufficient to stimulate 17,20-lyase activity maximally. Thus, it has not been clear how or if b 5 regulates human P450c17 activitiesin vivo. The use of microsomes from yeast engineered to express human P450c17, OR, or b 5 from inducible promoters permits the quantitative manipulation of each protein in a membrane environment that should simulate events in vivo. This permits greater experimental flexibility than the use of bicistronic plasmids (41Parikh A. Gillam E.M.J. Guengerich F.P. Nature Biotech. 1997; 15: 346-354Crossref Scopus (279) Google Scholar), fusion proteins (29Shet M.S. Fisher C.W. Arlotto M.P. Shackleton C.H.L. Holmans P.L. Martin-Wixtrom C.A. Saeki Y. Estabrook R.W. Arch. Biochem. Biophys. 1994; 311: 402-417Crossref PubMed Scopus (68) Google Scholar), or viral vectors (42Aoyama T. Nagata K. Yamazoe Y. Kato R. Matsunaga E. Gelboin H.V. Gonzalez F.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5425-5429Crossref PubMed Scopus (63) Google Scholar), and obviates concerns about the relevance of data from detergent-solubilized systems to in vivo systems. Titration experiments with purified human holo-b 5, apo-b 5, and cytochrome c showed that the stimulatory effect ofb 5 on 17,20-lyase activity is not mediated by electron transfer from b 5 and suggest thatb 5 exerts an allosteric effect on the P450c17·OR complex. This proposed mechanism could explain three observations from other laboratories. First, b 5facilitates electron transfer from OR to P450 3A4 only when all three proteins are premixed before adding NADPH and substrate, but not whenb 5 is premixed with P450 3A4 and added to OR, NADPH, and substrate in stop-flow experiments (24Yamazaki 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 (187) Google Scholar). These data suggested that the stimulatory action of b 5 on testosterone 6β-hydroxylation by P450 3A4 was an allosteric effect and was not mediated by an action of b 5 as an alternate electron donor (24Yamazaki 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 (187) Google Scholar). Second, b 5 is a more potent stimulator of 17,20-lyase activity when the abundance of OR is low, and this stimulation is quite sensitive to small changes in these low amounts of OR (10Kominami S. Ogawa N. Morimune R. Huang D.Y. Takemori S. J. Steroid Biochem. Mol. Biol. 1992; 42: 57-64Crossref PubMed Scopus (84) Google Scholar). Our results corroborate these studies and suggest that b 5 interacts primarily with the P450c17·OR complex and not with P450c17 alone. Third, the redox-active core 1 segment of porcine b 5 alone cannot augment the 17,20-lyase activity of human P450c17 (43Lee-Robichaud P. Kaderbhai M.A. Kaderbhai N. Wright J.N. Akhtar M. Biochem. J. 1997; 321: 857-863Crossref PubMed Scopus (44) Google Scholar), consistent with our findings that electron transfer from humanb 5 is not required to stimulate 17,20-lyase activity. Three conclusions about human physiology emerge from our analysis of the kinetics of human P450c17. First, human androgen biosynthesis proceeds predominantly through the pathway 17α-hydroxypregnenolone → DHEA → androstenedione, rather than through the pathway 17α-hydroxypregnenolone → 17α-hydroxyprogesterone → androstenedione. The pathway via DHEA predominates because the apparentK m for Δ4 17α-hydroxyprogesterone is about 10-fold higher and its V max is one-tenth as fast as the corresponding values for Δ517α-hydroxypregnenolone. Thus, the catalytic efficiencyV max/K m for the 17,20-lyase reaction is nearly 100-fold greater for Δ517α-hydroxypregnenolone than for Δ417α-hydroxyprogesterone. Second, significant androgen biosynthesis via the Δ4 pathway can only occur in the presence of very high Δ4 17α-hydroxyprogesterone concentrations, as found in untreated patients with 21-hydroxylase deficiency (44Miller W.L. Morel Y. Annu. Rev. Genet. 1989; 23: 371-393Crossref PubMed Scopus (110) Google Scholar). Third, considerable microsomal 17,20-lyase activity is found even in the complete absence of b 5; therefore, b 5 deficiency cannot cause a syndrome of complete 17,20-lyase deficiency (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar) as has been suggested (45Giordano S.J. Kaftory A. Steggles A.W. Hum. Genet. 1994; 93: 568-570Crossref PubMed Scopus (59) Google Scholar). The structural nature of the interaction of P450c17 with OR is not known, but the x-ray crystal structures of rat OR (46Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar) and P450-BMP (47Ravichandran K.G. Boddupalli S.S. Hasemann C.A. Peterson J.A. Deisenhofer J. Science. 1993; 261: 731-736Crossref PubMed Scopus (904) Google Scholar) provide useful clues. The redox-partner binding site for P450-BMP, a Type II (microsomal) P450, comprises the surface surrounding a depression in the “proximal” face of the protein that extends down to the face of the heme opposite the substrate-binding pocket (47Ravichandran K.G. Boddupalli S.S. Hasemann C.A. Peterson J.A. Deisenhofer J. Science. 1993; 261: 731-736Crossref PubMed Scopus (904) Google Scholar, 48Hasemann C.A. Kurumbail R.G. Boddupalli S.S. Peterson J.A. Deisenhofer J. Structure. 1995; 3: 41-62Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). This crevasse is lined on one side with positively charged residues from the J′ and K helices (in P450-BMP, lysines 325, 328, and 331) which appear to participate in electrostatic pairing with negatively charged residues in OR. Molecular modeling shows that human P450c17 has a similarly located crevasse of positively charged residues that interact with redox partners (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar). The electron-donating FMN moiety of rat OR also lies at the base of a concave cleft formed by the butterfly-shaped apposition of the FMN and FAD domains (46Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar). However, the FMN domain joins the remainder of the protein via a disordered, flexible hinge that must flex about 90° for the FMN moiety to extend out from the concave cleft of OR (46Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar) to approach the concave redox-partner binding site of P450c17 (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar). Because b 5 normally participates in redox reactions such as methemoglobin reduction (49Kuma F. Prough R.A. Masters B.S. Arch. Biochem. Biophys. 1976; 172: 600-607Crossref PubMed Scopus (45) Google Scholar) and stearyl-CoA desaturation (50Oshino N. Imai Y. Sato R. J. Biochem. (Tokyo). 1971; 69: 155-167Crossref PubMed Scopus (313) Google Scholar) and can serve as an alternate electron donor in some other P450 reactions (51Yamazaki H. Gillam E.M. Dong M.S. Johnson W.W. Guengerich F.P. Shimada T. Arch. Biochem. Biophys. 1997; 342: 329-337Crossref PubMed Scopus (129) Google Scholar), our demonstration thatb 5 serves a role as an allosteric facilitator of electron transfer from OR to P450c17 was unexpected. The binding of redox partners to P450c17 must transmit subtle changes to the substrate-binding pocket, as evidenced by the lowerK m values for Δ5 substrates in the presence of human OR (Table III) and by analogy to the altered regiospecificity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine oxygenation by P450 2D6 in the presence of OR (28Modi S. Gilham D.E. Sutcliffe M.J. Lian L.Y. Primrose W.U. Wolf C.R. Roberts G.C. Biochemistry. 1997; 36: 4461-4470Crossref PubMed Scopus (88) Google Scholar). Furthermore, the oxidative scission of the C17-C20 bond of 17α-hydroxypregnenolone appears to impose much more stringent constraints on the active-site topology of P450c17 than does the 17α-hydroxylase reaction. Therefore, we propose thatb 5 optimizes the geometry of the P450c17·OR complex for the more sensitive 17,20-lyase reaction perhaps by forming a ternary complex (Fig. 7). The structural core 2 domain of b 5 may be the region that stimulates 17,20-lyase activity, as core 2 adopts a similar conformation in the NMR structures of both isolated holo-b 5 (52Muskett F.W. Kelly G.P. Whitford D. J. Mol. Biol. 1996; 258: 172-189Crossref PubMed Scopus (47) Google Scholar) and apo-b 5(53Falzone C.J. Mayer M.R. Whiteman E.L. Moore C.D. Lecomte J.T. Biochemistry. 1996; 35: 6519-6526Crossref PubMed Scopus (81) Google Scholar), whereas the heme-binding core 1 domain is disordered in apo-b 5 (53Falzone C.J. Mayer M.R. Whiteman E.L. Moore C.D. Lecomte J.T. Biochemistry. 1996; 35: 6519-6526Crossref PubMed Scopus (81) Google Scholar). Furthermore, core 2 retains its overall topology during molecular dynamics simulations of apo-b 5, while core 1 loses secondary structure and exhibits conformational mobility (54Storch E.M. Daggett V. Biochemistry. 1996; 35: 11596-11604Crossref PubMed Scopus (31) Google Scholar). A role for b 5 as an allosteric effector protein is consistent with our observation that serine phosphorylation of P450c17 selectively increases 17,20-lyase activity (8Zhang L. Rodriguez H. Ohno S. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10619-10623Crossref PubMed Scopus (398) Google Scholar) and that mutations of arginine residues in the redox-partner binding site of human P450c17 cause isolated 17,20-lyase deficiency (23Geller D.H. Auchus R.J. Mendonça B.B. Miller W.L. Nature Genet. 1997; 17: 201-205Crossref PubMed Scopus (251) Google Scholar). The precise orientation of OR in the electron-donor docking region of P450c17 required to assemble the active oxygenating complex for the 17,20-lyase reaction is impaired by mutation of this surface and enhanced byb 5 or apo-b 5. Phosphorylation of P450c17 probably favors assembly of productive complexes so that electron transfer is more rapid and coupling efficiency is higher; however, the exact mechanism by which phosphorylated serine residues enhance 17,20-lyase activity is not yet known. A more detailed understanding of these complexes is essential for understanding the regulation of 17,20-lyase activity; this in turn may permit development of agents to inhibit this activity, which will aid in the treatment of sex steroid-dependent malignancies and disorders of androgen excess. We thank Dr. Denis Pompon for yeast strains W(B), W(hR), and W(BΔ) and for yeast vectors V10 and V60; Drs. Gregory Petsko and Ira Herskowitz for yeast strains W303A and W303B and for vector pYcDE2; Dr. C. Roland Wolf for antiserum to human OR; and Drs. Phillipe Urban and Gilles Truan for valuable discussions.
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