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• W2725904614 abstract "Cytochrome P450 (P450, CYP) 17A1 plays a critical role in steroid metabolism, catalyzing both the 17α-hydroxylation of pregnenolone and progesterone and the subsequent 17α,20-lyase reactions to form dehydroepiandrosterone (DHEA) and androstenedione (Andro), respectively, critical for generating glucocorticoids and androgens. Human P450 17A1 reaction rates examined are enhanced by the accessory protein cytochrome b5 (b5), but the exact role of b5 in P450 17A1-catalyzed reactions is unclear as are several details of these reactions. Here, we examined in detail the processivity of the 17α-hydroxylation and lyase steps. b5 did not enhance reaction rates by decreasing the koff rates of any of the steroids. Steroid binding to P450 17A1 was more complex than a simple two-state system. Pre-steady-state experiments indicated lag phases for Andro production from progesterone and for DHEA from pregnenolone, indicating a distributive character of the enzyme. However, we observed processivity in pregnenolone/DHEA pulse–chase experiments. (S)-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC50 values for (S)-orteronel were identical for blocking DHEA formation from pregnenolone and for 17α-hydroxylation, suggestive of processivity. Global kinetic modeling helped assign sets of rate constants for individual or groups of reactions, indicating that human P450 17A1 is an inherently distributive enzyme but that some processivity is present, i.e. some of the 17α-OH pregnenolone formed from pregnenolone did not dissociate from P450 17A1 before conversion to DHEA. Our results also suggest multiple conformations of P450 17A1, as previously proposed on the basis of NMR spectroscopy and X-ray crystallography. Cytochrome P450 (P450, CYP) 17A1 plays a critical role in steroid metabolism, catalyzing both the 17α-hydroxylation of pregnenolone and progesterone and the subsequent 17α,20-lyase reactions to form dehydroepiandrosterone (DHEA) and androstenedione (Andro), respectively, critical for generating glucocorticoids and androgens. Human P450 17A1 reaction rates examined are enhanced by the accessory protein cytochrome b5 (b5), but the exact role of b5 in P450 17A1-catalyzed reactions is unclear as are several details of these reactions. Here, we examined in detail the processivity of the 17α-hydroxylation and lyase steps. b5 did not enhance reaction rates by decreasing the koff rates of any of the steroids. Steroid binding to P450 17A1 was more complex than a simple two-state system. Pre-steady-state experiments indicated lag phases for Andro production from progesterone and for DHEA from pregnenolone, indicating a distributive character of the enzyme. However, we observed processivity in pregnenolone/DHEA pulse–chase experiments. (S)-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC50 values for (S)-orteronel were identical for blocking DHEA formation from pregnenolone and for 17α-hydroxylation, suggestive of processivity. Global kinetic modeling helped assign sets of rate constants for individual or groups of reactions, indicating that human P450 17A1 is an inherently distributive enzyme but that some processivity is present, i.e. some of the 17α-OH pregnenolone formed from pregnenolone did not dissociate from P450 17A1 before conversion to DHEA. Our results also suggest multiple conformations of P450 17A1, as previously proposed on the basis of NMR spectroscopy and X-ray crystallography. The cytochrome P450 (P450 or CYP) 3The abbreviations used are: P450 (or CYP), cytochrome P450; Andro, androstenedione; b5, cytochrome b5; compound I, formal FeO3+ oxidized form of a hemoprotein; DHEA, dehydroepiandrosterone; DLPC, l-α-1,2-dilauoryl-sn-glycero-3-phosphocholine; OH, hydroxy; SVD, singular value decomposition. enzymes collectively have the most diverse set of substrates, at least among oxidoreductases (1.Rendic S. Guengerich F.P. Survey of human oxidoreductases and cytochrome P450 enzymes involved in the metabolism of xenobiotic and natural chemicals.Chem. Res. Toxicol. 2015; 28: 38-42Crossref PubMed Scopus (243) Google Scholar). In particular, P450s are very important catalysts in the metabolism of steroids (human P450s 1B1, 7A1, 7B1, 8B1, 11A1, 11B1, 11B2, 17A1, 19A1, 21A2, 24A1, 27A1, 39A1, 46A1, and 51A1, plus some others involved in catabolism) and vitamins (human P450s 2R1, 24A1, 26A1, 26B1, 26C1, 27A1, 27B1, and 27C1) (2.Auchus R.J. Miller W.L. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 4th Ed. Springer, New York2015: 851-879Google Scholar, 3.Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 4th Ed. Springer, New York2015: 523-785Google Scholar). The steroidogenic P450s are particularly important, and homozygous or dual heterozygous deficiencies are often debilitating (2.Auchus R.J. Miller W.L. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 4th Ed. Springer, New York2015: 851-879Google Scholar, 4.Auchus R.J. Steroid 17-hydroxylase and 17,20-lyase deficiencies, genetic and pharmacologic.J. Steroid Biochem. Mol. Biol. 2017; 165: 71-78Crossref PubMed Scopus (104) Google Scholar). P450 enzymes also have major roles in the metabolism of exogenous (xenobiotic) chemicals, including drugs and carcinogens (3.Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 4th Ed. Springer, New York2015: 523-785Google Scholar), and some P450s oxidize both endogenous and xenobiotic substrates (5.Guengerich F.P. Intersection of roles of cytochrome P450 enzymes with xenobiotic and endogenous substrates. Relevance to toxicity and drug interactions.Chem. Res. Toxicol. 2017; 30: 2-12Crossref PubMed Scopus (85) Google Scholar). One of the steroidogenic P450s, P450 17A1, has a critical role in the production of androgens and also glucocorticoids (2.Auchus R.J. Miller W.L. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 4th Ed. Springer, New York2015: 851-879Google Scholar). At least 50 variants with P450 17A1 insufficiency have been identified clinically (4.Auchus R.J. Steroid 17-hydroxylase and 17,20-lyase deficiencies, genetic and pharmacologic.J. Steroid Biochem. Mol. Biol. 2017; 165: 71-78Crossref PubMed Scopus (104) Google Scholar, 6.DeVore N.M. Scott E.E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.Nature. 2012; 482: 116-119Crossref PubMed Scopus (251) Google Scholar). The enzyme catalyzes the two-step oxidation of progesterone to 17α-OH progesterone to androstenedione (Andro) and also the oxidation of pregnenolone to 17α-OH pregnenolone to dehydroepiandrosterone (DHEA) (Fig. 1). The second reaction in each series (Fig. 1A) is termed a “lyase” (or “desmolase”) reaction, in that cleavage of the 17,20 C–C bond occurs. Although these were first considered to be separate enzymatic activities, they are now known to be catalyzed by a single protein, P450 17A1 (7.Nakajin S. Shively J.E. Yuan P.M. Hall P.F. Microsomal cytochrome P-450 from neonatal pig testis: two enzymatic activities (17α-hydroxylase and C17,20-lyase) associated with one protein.Biochemistry. 1981; 20: 4037-4042Crossref PubMed Scopus (256) Google Scholar, 8.Zuber M.X. Simpson E.R. Waterman M.R. Expression of bovine 17α-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS-1) cells.Science. 1986; 234: 1258-1261Crossref PubMed Scopus (349) Google Scholar9.Chung B.C. Picado-Leonard J. Haniu M. Bienkowski M. Hall P.F. Shively J.E. Miller W.L. Cytochrome P450c17 (steroid 17α-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 407-411Crossref PubMed Scopus (411) Google Scholar). The presence of another hemoprotein, cytochrome b5 (b5), has been shown to greatly stimulate the lyase activity (10.Katagiri M. Suhara K. Shiroo M. Fujimura Y. Role of cytochrome b5 in the cytochrome P-450-mediated C21-steroid 17,20-lyase reaction.Biochem. Biophys. Res. Commun. 1982; 108: 379-384Crossref PubMed Scopus (54) Google Scholar11.Katagiri M. Kagawa N. Waterman M.R. The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (208) Google Scholar, 12.Lee-Robichaud P. Wright J.N. Akhtar M.E. Akhtar M. Modulation of the activity of human 17α-hydroxylase-17,20-lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications.Biochem. J. 1995; 308: 901-908Crossref PubMed Scopus (133) Google Scholar13.Estrada D.F. Skinner A.L. Laurence J.S. Scott E.E. Human cytochrome P450 17A1 conformational selection: modulation by ligand and cytochrome b5.J. Biol. Chem. 2014; 289: 14310-14320Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), at least in some species. The process is complicated by accompanying minor reactions (Fig. 1B). The minor products have all been identified in vitro (14.Yoshimoto F.K. Zhou Y. Peng H.M. Stidd D. Yoshimoto J.A. Sharma K.K. Matthew S. Auchus R.J. Minor activities and transition state properties of the human steroid hydroxylases cytochromes P450c17 and P450c21, from reactions observed with deuterium-labeled substrates.Biochemistry. 2012; 51: 7064-7077Crossref PubMed Scopus (30) Google Scholar, 15.Yoshimoto F.K. Auchus R.J. The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1).J. Steroid Biochem. Mol. Biol. 2015; 151: 52-65Crossref PubMed Scopus (64) Google Scholar16.Yoshimoto F.K. Gonzalez E. Auchus R.J. Guengerich F.P. Mechanism of 17α,20-lyase and new hydroxylation reactions of human cytochrome P450 17A1: 18O labeling and oxygen surrogate evidence for a role of a perferryl oxygen.J. Biol. Chem. 2016; 291: 17143-17164Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and 16α-OH progesterone has been measured in human serum (17.Turcu A.F. Rege J. Chomic R. Liu J. Nishimoto H.K. Else T. Moraitis A.G. Palapattu G.S. Rainey W.E. Auchus R.J. Profiles of 21-carbon steroids in 21-hydroxylase deficiency.J. Clin. Endocrinol. Metab. 2015; 100: 2283-2290Crossref PubMed Scopus (56) Google Scholar). A role for b5 in enhancing P450 catalytic activity was first shown in liver microsomes in 1971 (18.Estabrook R.W. Hildebrandt A.G. Baron J. Netter K.J. Leibman K. A new spectral intermediate associated with cytochrome P-450 function in liver microsomes.Biochem. Biophys. Res. Commun. 1971; 42: 132-139Crossref PubMed Scopus (169) Google Scholar, 19.Hildebrandt A. Estabrook R.W. Evidence for the participation of cytochrome b5 in hepatic microsomal mixed-function oxidation reactions.Arch. Biochem. Biophys. 1971; 143: 66-79Crossref PubMed Scopus (453) Google Scholar). In 1982, b5 was shown to stimulate the 17α-OH progesterone lyase activity of purified porcine testicular P450 17A1 3–4-fold (10.Katagiri M. Suhara K. Shiroo M. Fujimura Y. Role of cytochrome b5 in the cytochrome P-450-mediated C21-steroid 17,20-lyase reaction.Biochem. Biophys. Res. Commun. 1982; 108: 379-384Crossref PubMed Scopus (54) Google Scholar, 20.Onoda M. Hall P.F. Cytochrome b5 stimulates purified testicular microsomal cytochrome P-450 (C21 side-chain cleavage).Biochem. Biophys. Res. Commun. 1982; 108: 454-460Crossref PubMed Scopus (117) Google Scholar). Kominami et al. (21.Kominami S. Ogawa N. Morimune R. De-Ying H. Takemori S. The role of cytochrome b5 in adrenal microsomal steroidogenesis.J. Steroid Biochem. Mol. Biol. 1992; 42: 57-64Crossref PubMed Scopus (84) Google Scholar) also showed stimulation of guinea pig P450 17A1 by bovine b5, both the 17α-hydroxylation of progesterone and, to a fractionally larger extent, the 17α-OH progesterone lyase reaction. However, b5 was inhibitory at a b5/P450 17A1 ratio of >0.5 (21.Kominami S. Ogawa N. Morimune R. De-Ying H. Takemori S. The role of cytochrome b5 in adrenal microsomal steroidogenesis.J. Steroid Biochem. Mol. Biol. 1992; 42: 57-64Crossref PubMed Scopus (84) Google Scholar). In 1995, Katagiri et al. (11.Katagiri M. Kagawa N. Waterman M.R. The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (208) Google Scholar) reported that human b5 (added in a concentration equimolar to P450) stimulated several catalytic activities of human P450 17A1. Progesterone 17α-hydroxylation was not stimulated by b5, but pregnenolone 17α-hydroxylation was enhanced 2.5-fold; lyase activities toward both 17α-OH progesterone and 17α-OH pregnenolone were enhanced ∼10-fold by b5 (11.Katagiri M. Kagawa N. Waterman M.R. The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (208) Google Scholar). No qualitative difference was observed in the binding of 17α-OH pregnenolone to P450 17A1. In the same year (1995), Lee-Robichaud et al. (12.Lee-Robichaud P. Wright J.N. Akhtar M.E. Akhtar M. Modulation of the activity of human 17α-hydroxylase-17,20-lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications.Biochem. J. 1995; 308: 901-908Crossref PubMed Scopus (133) Google Scholar) also demonstrated stimulation of both porcine and human P450 17A1 activities by porcine b5, although the results differed from those of Waterman and co-workers (11.Katagiri M. Kagawa N. Waterman M.R. The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (208) Google Scholar). The catalytic efficiency of 17α-hydroxylation was increased 2-fold for pregnenolone but not for progesterone, as in the report of Waterman and co-workers (11.Katagiri M. Kagawa N. Waterman M.R. The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (208) Google Scholar). However, no lyase activity with either 17α-OH progesterone or 17α-OH pregnenolone (as substrate) could be detected in the absence of b5, but activities were seen in the presence of a 5-fold molar excess of b5 > P450 17A1 (12.Lee-Robichaud P. Wright J.N. Akhtar M.E. Akhtar M. Modulation of the activity of human 17α-hydroxylase-17,20-lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications.Biochem. J. 1995; 308: 901-908Crossref PubMed Scopus (133) Google Scholar). There are several enigmas about P450 17A1. One is the exact role of b5, which NMR studies indicate occupies the same site as and competes with the obligate electron donor NADPH-P450 reductase (13.Estrada D.F. Skinner A.L. Laurence J.S. Scott E.E. Human cytochrome P450 17A1 conformational selection: modulation by ligand and cytochrome b5.J. Biol. Chem. 2014; 289: 14310-14320Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 22.Estrada D.F. Laurence J.S. Scott E.E. Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.J. Biol. Chem. 2013; 288: 17008-17018Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). b5 does not appear to transfer electrons to P450 at any point in the catalytic cycle (23.Auchus R.J. Lee T.C. Miller W.L. Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer.J. Biol. Chem. 1998; 273: 3158-3165Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 24.Lee-Robichaud P. Akhtar M.E. Akhtar M. Control of androgen biosynthesis in the human through the interaction of Arg347 and Arg358 of CYP17 with cytochrome b5.Biochem. J. 1998; 332: 293-296Crossref PubMed Scopus (44) Google Scholar). Another controversy is whether a ferric peroxide (FeO2−) or compound I (perferryl oxygen; FeO3+) species is involved in the lyase reaction (15.Yoshimoto F.K. Auchus R.J. The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1).J. Steroid Biochem. Mol. Biol. 2015; 151: 52-65Crossref PubMed Scopus (64) Google Scholar, 16.Yoshimoto F.K. Gonzalez E. Auchus R.J. Guengerich F.P. Mechanism of 17α,20-lyase and new hydroxylation reactions of human cytochrome P450 17A1: 18O labeling and oxygen surrogate evidence for a role of a perferryl oxygen.J. Biol. Chem. 2016; 291: 17143-17164Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 25.Lee-Robichaud P. Shyadehi A.Z. Wright J.N. Akhtar M.E. Akhtar M. Mechanistic kinship between hydroxylation and desaturation reactions: acyl-carbon bond cleavage promoted by pig and human CYP17 (P-45017α; 17α-hydroxylase-17,20-lyase).Biochemistry. 1995; 34: 14104-14113Crossref PubMed Scopus (63) Google Scholar, 26.Akhtar M. Wright J.N. Lee-Robichaud P. A review of mechanistic studies on aromatase (CYP19) and 17α-hydroxylase-17,20-lyase (CYP17).J. Steroid Biochem. Mol. Biol. 2011; 125: 2-12Crossref PubMed Scopus (94) Google Scholar27.Mak P.J. Gregory M.C. Denisov I.G. Sligar S.G. Kincaid J.R. Unveiling the crucial intermediates in androgen production.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 15856-15861Crossref PubMed Scopus (54) Google Scholar). One of the issues regarding the two-step, net four-electron oxidation (Fig. 1A) is the processivity of the overall reaction. In a processive reaction, the 17α-OH steroid product remains bound to the enzyme for the succeeding lyase reaction, but in a distributive reaction, the 17α-OH product dissociates and then re-binds for the lyase reaction (Fig. 1A). A variety of reports on the topic of processivity have appeared, with varying conclusions. On the basis of work in human embryonic kidney (HEK-293) cells, Soucy and Luu-The (28.Soucy P. Luu-The V. Conversion of pregnenolone to DHEA by human 17α-hydroxylase/17,20-lyase (P450c17). Evidence that DHEA is produced from the released intermediate, 17α-hydroxypregnenolone.Eur. J. Biochem. 2000; 267: 3243-3247Crossref PubMed Scopus (30) Google Scholar) concluded that this is a distributive sequence of reactions. However, studies with rat, guinea pig, and bovine P450 17A1 enzymes led to conclusions that the two steps are processive (29.Higuchi A. Kominami S. Takemori S. Kinetic control of steroidogenesis by steroid concentration in guinea pig adrenal microsomes.Biochim. Biophys. Acta. 1991; 1084: 240-246Crossref PubMed Scopus (28) Google Scholar30.Kühn-Velten W.N. Bunse T. Förster M.E. Enzyme kinetic and inhibition analyses of cytochrome P450XVII, a protein with a bifunctional catalytic site: quantification of effective substrate concentrations at the active site and their significance for intrinsic control of the hydroxylase/lyase reaction sequence.J. Biol. Chem. 1991; 266: 6291-6301Abstract Full Text PDF PubMed Google Scholar, 31.Yamazaki T. Marumoto T. Kominami S. Ishimura K. Yamamoto A. Takemori S. Kinetic studies on androstenedione production in ovarian microsomes from immature rats.Biochim. Biophys. Acta. 1992; 1125: 335-340Crossref PubMed Scopus (9) Google Scholar, 32.Tagashira H. Kominami S. Takemori S. Kinetic studies of cytochrome P45017α,lyase dependent androstenedione formation from progesterone.Biochemistry. 1995; 34: 10939-10945Crossref PubMed Scopus (25) Google Scholar33.Yamazaki T. Ohno T. Sakaki T. Akiyoshi-Shibata M. Yabusaki Y. Imai T. Kominami S. Kinetic analysis of successive reactions catalyzed by bovine cytochrome P45017α,lyase.Biochemistry. 1998; 37: 2800-2806Crossref PubMed Scopus (43) Google Scholar). These studies involved a number of approaches and designs, but none included b5, which had already been shown to stimulate the lyase reaction (10.Katagiri M. Suhara K. Shiroo M. Fujimura Y. Role of cytochrome b5 in the cytochrome P-450-mediated C21-steroid 17,20-lyase reaction.Biochem. Biophys. Res. Commun. 1982; 108: 379-384Crossref PubMed Scopus (54) Google Scholar, 11.Katagiri M. Kagawa N. Waterman M.R. The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (208) Google Scholar). Our own work with zebrafish P450 17A1 and b5 indicated that the reactions were distributive when progesterone was the substrate and more processive when pregnenolone was the substrate, although only a relatively weak effect of zebrafish or human b5 is seen in that system (34.Pallan P.S. Nagy L.D. Lei L. Gonzalez E. Kramlinger V.M. Azumaya C.M. Wawrzak Z. Waterman M.R. Guengerich F.P. Egli M. Structural and kinetic basis of steroid 17α,20-lyase activity in teleost fish cytochrome P450 17A1 and its absence in cytochrome P450 17A2.J. Biol. Chem. 2015; 290: 3248-3268Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The matter of processivity is important for several reasons. One is that 17α-OH progesterone and 17α-OH pregnenolone (formed following 2-electron reduction of 17α-OH progesterone) serve as precursors to glucocorticoids (2.Auchus R.J. Miller W.L. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 4th Ed. Springer, New York2015: 851-879Google Scholar, 3.Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 4th Ed. Springer, New York2015: 523-785Google Scholar). Another issue is that prostate cancer is androgen-stimulated (35.Huggins C. Stevens R.E. The effect of castration on benign hypertrophy of the prostate in man.J. Urol. 1940; 107 (2017 10.1016/S0022-5347(17)71453-X): S44-S75Google Scholar), and P450 17A1 is a major drug target for therapy (6.DeVore N.M. Scott E.E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.Nature. 2012; 482: 116-119Crossref PubMed Scopus (251) Google Scholar, 36.Bonomo S. Hansen C.H. Petrunak E.M. Scott E.E. Styrishave B. Jørgensen F.S. Olsen L. Promising tools in prostate cancer research: selective non-steroidal cytochrome P450 17A1 inhibitors.Sci. Rep. 2016; 6: 29468Crossref PubMed Scopus (34) Google Scholar37.Yin L. Hu Q. CYP17 inhibitors–abiraterone, C17,20-lyase inhibitors and multi-targeting agents.Nat. Rev. Urol. 2014; 11: 32-42Crossref PubMed Scopus (109) Google Scholar, 38.Toren P.J. Kim S. Pham S. Mangalji A. Adomat H. Guns E.S. Zoubeidi A. Moore W. Gleave M.E. Anticancer activity of a novel selective CYP17A1 inhibitor in preclinical models of castrate-resistant prostate cancer.Mol. Cancer Ther. 2015; 14: 59-69Crossref PubMed Scopus (74) Google Scholar39.Njar V.C. Brodie A.M. Discovery and development of galeterone (TOK-001 or VN/124–1) for the treatment of all stages of prostate cancer.J. Med. Chem. 2015; 58: 2077-2087Crossref PubMed Scopus (148) Google Scholar). A goal is the selective inhibition of the androgen-producing lyase reaction, preserving the ability to generate 17α-OH steroids as precursors for biosynthesis of glucocorticosteroids and avoiding adverse effects caused by insufficient glucocorticoid and excess mineralocorticoid production. Some selectivity for lyase > 17α-hydroxylation activity has been reported for abiraterone (36.Bonomo S. Hansen C.H. Petrunak E.M. Scott E.E. Styrishave B. Jørgensen F.S. Olsen L. Promising tools in prostate cancer research: selective non-steroidal cytochrome P450 17A1 inhibitors.Sci. Rep. 2016; 6: 29468Crossref PubMed Scopus (34) Google Scholar), orteronel (Takeda TAK-700) (40.Hara T. Kouno J. Kaku T. Takeuchi T. Kusaka M. Tasaka A. Yamaoka M. Effect of a novel 17,20-lyase inhibitor, orteronel (TAK-700), on androgen synthesis in male rats.J. Steroid Biochem. Mol. Biol. 2013; 134: 80-91Crossref PubMed Scopus (29) Google Scholar), the secosteroid (S)-seviteronel (VT-464) (41.Rafferty S.W. Eisner J.R. Moore W.R. Schotzinger R.J. Hoekstra W.J. Highly-selective 4-(1,2,3-triazole)-based P450c17α 17,20-lyase inhibitors.Bioorg. Med. Chem. Lett. 2014; 24: 2444-2447Crossref PubMed Scopus (46) Google Scholar), and two other non-steroidal pyridine derivatives (36.Bonomo S. Hansen C.H. Petrunak E.M. Scott E.E. Styrishave B. Jørgensen F.S. Olsen L. Promising tools in prostate cancer research: selective non-steroidal cytochrome P450 17A1 inhibitors.Sci. Rep. 2016; 6: 29468Crossref PubMed Scopus (34) Google Scholar). X-ray crystal structures of human P450 bound to abiraterone (6.DeVore N.M. Scott E.E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.Nature. 2012; 482: 116-119Crossref PubMed Scopus (251) Google Scholar) and to both enantiomers of orteronel (42.Petrunak E.M. Rogers S.A. Aubé J. Scott E.E. Structural and functional evaluation of clinically-relevant inhibitors of steroidogenic cytochrome P450 17A1 (CYP17A1).Drug Metab. Dispos. 2017; 45: 635-645Crossref PubMed Scopus (24) Google Scholar) have been published by Scott and co-workers (42.Petrunak E.M. Rogers S.A. Aubé J. Scott E.E. Structural and functional evaluation of clinically-relevant inhibitors of steroidogenic cytochrome P450 17A1 (CYP17A1).Drug Metab. Dispos. 2017; 45: 635-645Crossref PubMed Scopus (24) Google Scholar). A completely processive reaction should not have differential sensitivities to a single inhibitor. Even in a dissociative mechanism, the question arises as to how a drug can occupy a single site on the enzyme and have different Ki values for two reactions (42.Petrunak E.M. Rogers S.A. Aubé J. Scott E.E. Structural and functional evaluation of clinically-relevant inhibitors of steroidogenic cytochrome P450 17A1 (CYP17A1).Drug Metab. Dispos. 2017; 45: 635-645Crossref PubMed Scopus (24) Google Scholar, 43.Yadav R. Petrunak E.M. Estrada D.F. Scott E.E. Structural insights into the function of steroidogenic cytochrome P450 17A1.Mol. Cell Endocrinol. 2017; 441: 68-75Crossref PubMed Scopus (20) Google Scholar). For the above reasons, we examined the kinetics of binding of human P450 17A1 to its primary physiological substrates and products and also an enantiomer of orteronel. We also utilized pre-steady-state reaction kinetics and global data analysis to develop a model of the human P450 17A1 reactions. These studies began with steady-state reaction kinetics, which included an analysis of the effect of b5 (Fig. 2 and %Table 1). Some of the lack of ideal curve fitting may be due to the formation of additional products (Fig. 1B) (15.Yoshimoto F.K. Auchus R.J. The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1).J. Steroid Biochem. Mol. Biol. 2015; 151: 52-65Crossref PubMed Scopus (64) Google Scholar, 16.Yoshimoto F.K. Gonzalez E. Auchus R.J. Guengerich F.P. Mechanism of 17α,20-lyase and new hydroxylation reactions of human cytochrome P450 17A1: 18O labeling and oxygen surrogate evidence for a role of a perferryl oxygen.J. Biol. Chem. 2016; 291: 17143-17164Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), which were not included here for the sake of simplification. The efficient conversion of 17α-OH pregnenolone to DHEA confounds the results for formation of 17α-OH pregnenolone from pregnenolone (Fig. 2A), but the lyase reaction (using 17α-OH pregnenolone as the starting substrate (Fig. 2B)) was more straightforward. The decrease in apparent rates of DHEA formation in the presence of b5 (Fig. 2B) may be the result of facile 16-hydroxylation of DHEA (16.Yoshimoto F.K. Gonzalez E. Auchus R.J. Guengerich F.P. Mechanism of 17α,20-lyase and new hydroxylation reactions of human cytochrome P450 17A1: 18O labeling and oxygen surrogate evidence for a role of a perferryl oxygen.J. Biol. Chem. 2016; 291: 17143-17164Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Our kcat and Km values are similar to those of Lee-Robichaud et al. (12.Lee-Robichaud P. Wright J.N. Akhtar M.E. Akhtar M. Modulation of the activity of human 17α-hydroxylase-17,20-lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications.Biochem. J. 1995; 308: 901-908Crossref PubMed Scopus (133) Google Scholar), and the kcat values are as high or higher than reported for any human P450 17A1 preparations by others (11.Katagiri M. Kagawa N. Waterman M.R. The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (208) Google Scholar, 44.Petrunak E.M. DeVore N.M. Porubsky P.R. Scott E.E. Structures of human steroidogenic cytochrome P450 17A1 with substrates.J. Biol. Chem. 2014; 289: 32952-32964Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar45.Gregory M.C. Denisov I.G. Grinkova Y.V. Khatri Y. Sligar S.G. Kinetic solvent isotope effect in human P450 CYP17A1-mediated androgen formation: evidence for a reactive peroxoanion intermediate.J. Am. Chem. Soc. 2013; 135: 16245-16247Crossref PubMed Scopus (50) Google Scholar, 46.Khatri Y. Gregory M.C. Grinkova Y.V. Denisov I.G. Sligar S.G. Active site proton delivery and the lyase activity of human CYP17A1.Biochem. Biophys. Res. Commun. 2014; 443: 179-184Crossref PubMed Scopus (48) Google Scholar, 47.Peng H.M. Im S.C. Pearl N.M. Turcu A.F. Rege J. Waskell L. Auchus R.J. Cytochrome b5 activates the 17,20-lyase activity of human cytochrome P450 17A1 by increasing the coupling of NADPH consumption to androgen production.Biochemistry. 2016; 55: 4356-4365Crossref PubMed Scopus (33) Google Scholar48.Duggal R. Liu Y. Gregory M.C. Denisov I.G. Kincaid J.R. Sligar S.G. Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis.Biochem. Biophys. Res. Commun. 2016; 477: 202-208Crossref PubMed Scopus (24) Google Scholar).Table 1Steady-state parameters for human P450 17A1 reactionsSubstrateReactionkcatKmkcat/Km−b5+b5−b5+b5−b5+b5+/− b5 ratios−1μmμm−1 s−1Pregnenolone17α-Hydroxylation0.0037±0.00050.11±0.010.5±0.31.1±0.20.0074±0.00440.10±0.0214LyaseaThe rate was too low to accurately measure the steady-state kinetic parameters (see Fig. 6A).0.045±0.003aThe rate was too low to accurately measure the steady-state kinetic parameters (se" @default.
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• W2725904614 title "Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1" @default.
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