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Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1.
Gonzalez E, Guengerich FP
(2017) J Biol Chem 292: 13168-13185
MeSH Terms: 17-alpha-Hydroxypregnenolone, Androstenedione, Animals, Binding Sites, Biocatalysis, Cytochrome P-450 Enzyme Inhibitors, Cytochromes b5, Dehydroepiandrosterone, Humans, Imidazoles, Kinetics, Ligands, Models, Molecular, NADPH-Ferrihemoprotein Reductase, Naphthalenes, Oxidation-Reduction, Pregnenolone, Progesterone, Protein Conformation, Rats, Recombinant Proteins, Stereoisomerism, Steroid 17-alpha-Hydroxylase
Show Abstract · Added March 14, 2018
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 (), but the exact role of 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. did not enhance reaction rates by decreasing the 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. ()-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC values for ()-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, 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.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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23 MeSH Terms
Kinetic analysis of the three-step steroid aromatase reaction of human cytochrome P450 19A1.
Sohl CD, Guengerich FP
(2010) J Biol Chem 285: 17734-43
MeSH Terms: Amino Acid Sequence, Androstenedione, Animals, Aromatase, Cytochrome P-450 Enzyme System, Estrone, Humans, Iron, Kinetics, Models, Chemical, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary, Rats, Steroids
Show Abstract · Added March 7, 2014
Cytochrome P450 19A1 (P450 19A1), the aromatase, catalyzes the conversion of androgens to estrogens through a sequential three-step reaction, generating 19-hydroxy and 19-aldehyde intermediates en route to the product estrogen. A procedure for the heterologous expression and purification of P450 19A1 in Escherichia coli was developed (k(cat) of 0.06 s(-1) for the conversion of androstenedione to estrone). Binding of the substrate and intermediates show low micromolar dissociation constants and are at least two-step processes. Rates of reduction of the iron were fast in the presence of substrate, either intermediate, or product. P450 19A1 is a distributive rather than a processive enzyme, with the sequential reaction allowing free dissociation of the intermediates as revealed by pulse-chase experiments. Conversion of androstenedione to estrone (under single turnover conditions) generated a progress curve showing changes in the concentrations of the substrate, intermediates, and product. A minimal kinetic model containing the individual rate constants for the steps in P450 19A1 catalysis was developed to globally fit the time course of the overall reaction, the dissociation constants, the two-step ligand binding, the distributive character, the iron-reduction rates, and the steady-state conversion of the 19-hydroxy androstenedione and 19-aldehyde androstenedione intermediates to estrone.
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15 MeSH Terms
The ferrous-oxy complex of human aromatase.
Grinkova YV, Denisov IG, Waterman MR, Arase M, Kagawa N, Sligar SG
(2008) Biochem Biophys Res Commun 372: 379-82
MeSH Terms: Androstenedione, Aromatase, Humans, Iron, Lipid Bilayers, Nanostructures, Oxidation-Reduction, Phospholipids
Show Abstract · Added February 12, 2015
In this communication, we document the self-assembly of heterologously expressed truncated human aromatase (CYP19) into nanometer scale phospholipids bilayers (Nanodiscs). The resulting P450 CYP19 preparation is stable and can tightly associate with the substrate androstenedione to form a nearly complete high-spin ferric protein. Ferrous CYP19 in Nanodiscs was mixed anaerobically in a rapid-scan stopped-flow with atmospheric dioxygen and the formation of the ferrous-oxy complex observed. First order decay of the oxy-complex to release superoxide and regenerate the ferric enzyme was monitored kinetically. Surprisingly, the ferrous-oxy complex of aromatase is more stable than that of hepatic CYP3A4, opening the path to precisely determine the biochemical and biophysical properties of the reaction cycle intermediates in this important human drug target.
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8 MeSH Terms
Raman evidence for specific substrate-induced structural changes in the heme pocket of human cytochrome P450 aromatase during the three consecutive oxygen activation steps.
Tosha T, Kagawa N, Ohta T, Yoshioka S, Waterman MR, Kitagawa T
(2006) Biochemistry 45: 5631-40
MeSH Terms: Androstenedione, Aromatase, Carbon Monoxide, Ferrous Compounds, Heme, Humans, Models, Chemical, Oxygen, Spectrophotometry, Spectrum Analysis, Raman
Show Abstract · Added February 12, 2015
Specific substrate-induced structural changes in the heme pocket are proposed for human cytochrome P450 aromatase (P450arom) which undergoes three consecutive oxygen activation steps. We have experimentally investigated this heme environment by resonance Raman spectra of both substrate-free and substrate-bound forms of the purified enzyme. The Fe-CO stretching mode (nu(Fe)(-)(CO)) of the CO complex and Fe(3+)-S stretching mode (nu(Fe)(-)(S)) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The nu(Fe)(-)(CO) mode was upshifted from 477 to 485 and to 490 cm(-)(1) by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas nu(Fe)(-)(CO) was not observed for P450arom with 19-hydroxyandrostenedione, a substrate for the second step, indicating that the heme distal site is very flexible and changes its structure depending on the substrate. The 19-aldehyde-androstenedione binding could reduce the electron donation from the axial thiolate, which was evident from the low-frequency shift of nu(Fe)(-)(S) by 5 cm(-)(1) compared to that of androstenedione-bound P450arom. Changes in the environment in the heme distal site and the reduced electron donation from the axial thiolate upon 19-aldehyde-androstenedione binding might stabilize the ferric peroxo species, an active intermediate for the third step, with the suppression of the formation of compound I (Fe(4+)=O porphyrin(+)(*)) that is the active species for the first and second steps. We, therefore, propose that the substrates can regulate the formation of alternative reaction intermediates by modulating the structure on both the heme distal and proximal sites in P450arom.
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10 MeSH Terms
ErbB receptor signaling and therapeutic resistance to aromatase inhibitors.
Shin I, Miller T, Arteaga CL
(2006) Clin Cancer Res 12: 1008s-1012s
MeSH Terms: Acetyltransferases, Androstenedione, Aromatase Inhibitors, Breast Neoplasms, Carrier Proteins, Cell Line, Tumor, Corticosterone, Drug Resistance, Neoplasm, Female, Histone Acetyltransferases, Humans, Letrozole, Nitriles, Nuclear Receptor Coactivator 3, Oncogene Proteins, Promoter Regions, Genetic, Receptor, ErbB-2, Receptors, Estrogen, Signal Transduction, Trans-Activators, Transcriptional Activation, Triazoles
Show Abstract · Added March 5, 2014
We have investigated the effect of HER-2 overexpression on resistance to the aromatase inhibitor letrozole in MCF-7 breast cancer cells stably expressing cellular aromatase (MCF-7/CA). MCF-7/CA cells overexpressing HER-2 showed a >2-fold increase in estrogen receptor (ER)-mediated transcriptional reporter activity upon treatment with androstenedione compared with vector-only control MCF-7/CA cells. Co-treatment with letrozole did not abrogate androstenedione-induced transcription and cell proliferation in HER-2-overexpressing cells. Chromatin immunoprecipitation assays using cross-linked protein-DNA from MCF-7/CA/HER-2 cells indicated ligand-independent association of the ERalpha coactivators AIB-1 and CBP to the promoter region of the estrogen-responsive pS2 gene. Upon treatment with androstenedione, there were increased associations of AIB1 and CBP with the pS2 promoter in the HER-2-overexpressing compared with control MCF-7/CA cells. These results suggest that ligand-independent recruitment of coactivator complexes to estrogen-responsive promoters as a result of HER-2 overexpression may play a role in the development of letrozole resistance.
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22 MeSH Terms
Characterization of stable human aromatase expressed in E. coli.
Kagawa N, Hori H, Waterman MR, Yoshioka S
(2004) Steroids 69: 235-43
MeSH Terms: Amino Acid Sequence, Androstenedione, Arginine, Aromatase, Aromatase Inhibitors, Cell Membrane, Chaperonin 10, Chaperonin 60, Chloramphenicol, Cysteine, Electron Spin Resonance Spectroscopy, Enzyme Stability, Escherichia coli, Estradiol, Estrone, Fadrozole, Gene Expression, Humans, Letrozole, Molecular Sequence Data, Nitriles, Polymorphism, Genetic, Protein Structure, Secondary, Protein Synthesis Inhibitors, Recombinant Proteins, Solubility, Testosterone, Triazoles
Show Abstract · Added February 12, 2015
Aromatase (P450arom, CYP19) catalyzes the aromatization reaction that converts androgens to estrogens. Although human P450arom has been readily purified from placenta, its hydrophobic properties and instability has hampered detailed characterization. Utilizing a N-terminal replacement (MARQSFGRGKL, derived from CYP2C11), we successfully modified this unstable enzyme into stable forms. Based on a known polymorphism, we created two constructs, NmA264C and NmA264R having cysteine or arginine at position 264. The recombinant P450arom NmA264R was expressed in Escherichia coli (350-400 nmol/L culture) primarily by coexpression with molecular chaperones GroES/GroEL while NmA264C was expressed (240 nmol/L culture) only in the presence of chloramphenicol. Although NmA264C was recovered only in the membrane fraction, approximately 14% of NmA264R was recovered in the cytosolic fraction, suggesting that NmA264R is more hydrophilic than NmA264C. NmA264R was highly purified to the specific content 13.6 nmol P450/mg protein. Purified P450arom NmA264R converted androstenedione to estrone with Vmax 12.4 nmol/(min nmol) and Km) 0.26 microM, and testosterone to estradiol with Vmax 52.2 nmol/(min nmol) and Km 10.9 microM. Because of the increased stability of NmA264R, we could unambiguously determine properties of human P450arom by optical and electron paramagnetic resonance spectroscopy. The purified protein was a typical low-spin form, which was converted to a high-spin form when androstenedione was added. The rhombicity of substrate-bound forms was higher than that reported for other P450s, an interesting characteristic of human P450arom. The highly stable and active P450arom NmA264R sets the stope for detailed structure/function analyses of this important member of the P450 superfamily.
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28 MeSH Terms
Premature pubarche in girls is associated with functional adrenal but not ovarian hyperandrogenism.
Mathew RP, Najjar JL, Lorenz RA, Mayes DE, Russell WE
(2002) J Pediatr 141: 91-8
MeSH Terms: 17-alpha-Hydroxyprogesterone, Adrenal Cortex, Androstenedione, Biomarkers, Case-Control Studies, Child, Child, Preschool, Dehydroepiandrosterone, Estradiol, Female, Hirsutism, Humans, Hyperandrogenism, Immunoenzyme Techniques, Ovary, Statistics, Nonparametric, Steroids
Show Abstract · Added December 10, 2013
OBJECTIVE - We hypothesized that there would be evidence of functional ovarian hyperandrogenism in girls with premature pubarche (PP) at diagnosis.
METHODS - White girls <8 years of age and black girls <6 years with PP (n = 15) were studied. Prepubertal girls (n = 13; 5.3-10.9 years) and early pubertal girls (n = 8) served as control subjects. The biochemical marker for functional ovarian hyperandrogenism was the 17-hydroxyprogesterone (17-OHP), androstenedione (AD), and estradiol (E2) response to subcutaneous leuprolide during adrenal suppression with dexamethasone. This was studied in girls with PP and in control subjects.
RESULTS - ACTH stimulated 17-hydroxypregnenolone (17-OH Preg), dehydroepiandrosterone (DHEA), and AD levels, and 17-OH Preg:17-OHP and DHEA:AD ratios were significantly higher in girls with PP than in prepubertal control subjects (n = 18) (P < or =.003). The ovarian response to leuprolide stimulation was comparable in girls with PP and prepubertal control subjects, but the response in prepubertal study subjects was significantly lower than in pubertal control subjects (P =.016 for Delta17-OHP, P =.001 for DeltaAD, and P =.026 for DeltaE2).
CONCLUSIONS - Contrary to the hypothesis, PP in girls was not associated with prepubertal evidence of ovarian hyperandrogenism but was associated with functional adrenal hyperandrogenism.
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17 MeSH Terms
Androgens and masculinization of genitalia in the spotted hyaena (Crocuta crocuta). 1. Urogenital morphology and placental androgen production during fetal life.
Licht P, Hayes T, Tsai P, Cunha G, Kim H, Golbus M, Hayward S, Martin MC, Jaffe RB, Glickman SE
(1998) J Reprod Fertil 113: 105-16
MeSH Terms: Androgens, Androstenedione, Animals, Carnivora, Clitoris, Female, Gestational Age, Male, Mesonephros, Mullerian Ducts, Ovary, Placenta, Sex Differentiation, Testis, Urethra, Urogenital System, Wolffian Ducts
Show Abstract · Added May 27, 2014
According to common understanding of sexual differentiation, the formation and development of a penile clitoris in female spotted hyaenas requires the presence of naturally circulating androgens during fetal life. The purpose of the present study was to determine potential source(s) of such fetal androgens by investigating the timing of urogenital development and placental production of androgen during early and mid-gestation. Fetuses determined to be female by molecular techniques (lack of SRY gene) at days 33 and 48 of gestation had undifferentiated gonads, but the clitoris was already 'masculinized' and was generally similar to the phallus of a 50-day-old male fetus. Wolffian and Müllerian ducts terminated at the urogenital sinus in both sexes and a urethra was present along the entire length of the clitoris and penis. The adrenal gland was large and histologically differentiated at 33 days. Steroid gradients across the uterus (a drop in delta 4-androstenedione, with increases in oestrogen and androgen), and high androstenedione in ovarian veins indicated that ovarian androstenedione was metabolized and secreted as testosterone by the placenta throughout gestation. In vitro, whole or homogenized placentae at days 48 and 58 of gestation (110 days total) metabolized radiolabelled androstenedione into testosterone and oestradiol; the specific enzymatic activity of early placental tissues was higher than at later stages. A human placental homogenate had higher aromatase activity but did not produce testosterone unless aromatase was inhibited. Infusion of labelled androstenedione into the uterine arteries of hyaenas demonstrated the conversion of this substrate into testosterone and oestradiol and their secretion into the fetal circulation. Evidently, androgen is produced by the placenta and secreted into the fetal circulation from early in pregnancy when masculinization is first evident, before differentiation of the fetal ovary.
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17 MeSH Terms
The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.
Katagiri M, Kagawa N, Waterman MR
(1995) Arch Biochem Biophys 317: 343-7
MeSH Terms: 17-alpha-Hydroxypregnenolone, 17-alpha-Hydroxyprogesterone, Aldehyde-Lyases, Androgens, Androstenedione, Cell Membrane, Cytochrome P-450 Enzyme System, Cytochromes b5, Dehydroepiandrosterone, Escherichia coli, Humans, Hydroxyprogesterones, Kinetics, Pregnenolone, Progesterone, Recombinant Proteins, Steroid 17-alpha-Hydroxylase
Show Abstract · Added February 12, 2015
Human cytochrome b5 has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P450c17. It enhances the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17 alpha-hydroxyprogesterone to androstenedione by at least 10-fold. This latter activity is virtually undetectable in the absence of cytochrome b5. Other activities catalyzed by P450c17 include 17 alpha-hydroxylation of progesterone and pregnenolone and are much less influenced by cytochrome b5. The conversion of pregnenolone to 17 alpha-hydroxypregnenolone is increased by 2-fold, while that of progesterone to 17 alpha-hydroxyprogesterone is unchanged. These studies using purified systems suggest that cytochrome b5 plays a role in regulating the activities of P450c17 to optimize the balance between sex hormone synthesis and glucocorticoid synthesis. In particular, they indicate that in human testes which contains a high b5/P450 ratio, 17 alpha-hydroxyprogesterone can serve as an intermediate in testosterone production, rather than being a dead-end product, or stated another way, because of the relatively high concentration of cytochrome b5 in the human testis, both the delta 4 and the delta 5 steroidogenic pathways can lead to testosterone production.
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17 MeSH Terms
Effects of ACTH on steroidogenesis in bovine adrenocortical cells in primary culture--increased secretion of 17 alpha-hydroxylated steroids associated with a refractoriness in total steroid output.
Kramer RE, McCarthy JL, Simpson ER, Waterman MR
(1983) J Steroid Biochem 18: 715-23
MeSH Terms: Adrenal Cortex, Adrenal Cortex Hormones, Adrenocorticotropic Hormone, Androstenedione, Animals, Cattle, Cells, Cultured, Corticosterone, Dehydroepiandrosterone, Hydrocortisone, Hydroxycorticosteroids, Kinetics, Progesterone
Show Abstract · Added February 12, 2015
The long-term effects of ACTH on steroidogenesis in bovine adrenocortical cells maintained in primary culture have been investigated. Cells in monolayer culture were incubated in the presence or absence of ACTH for up to 72 h, and the steroid content of the incubation medium was assayed at 12 h intervals. During the first 12 h, adrenocortical cells incubated in the presence of ACTH (10(-9) M and 10(-6) M) produced substantially more cortisol and corticosterone than did cells incubated in the absence of ACTH. The production of steroidogenic intermediates such as pregnenolone, progesterone, and 17 alpha-hydroxypregnenolone, as well as 17 alpha-hydroxyprogesterone, 11-deoxycortisol, and 11-deoxycorticosterone also was increased by short-term (12 h) treatment with ACTH. Thereafter, corticosteroid production by cells incubated in the continued presence of ACTH decreased in a time and concentration dependent fashion. The maximal rate of cortisol production by cells incubated in the presence of ACTH (10(-9) M and 10(-6) M) for 72 h was only one third that of cells incubated in the presence of ACTH for 12 h. More dramatically, by 36 h, corticosterone secretion by cells incubated in the presence of ACTH (10(-6) M) declined to less than 20% of that of nontreated cells, and the production of 11-deoxycorticosterone was no longer detectable. ACTH also induced refractoriness in the production of other C21-steroids (pregnenolone, progesterone, 17 alpha-hydroxypregnenolone, 17 alpha-hydroxyprogesterone, and 11-deoxycortisol) as well as of C19-steroids (dehydroepiandrosterone, androstenedione, and 11 beta-hydroxyandrostenedione). The ACTH-induced refractoriness in the production of C21-steroids lacking a 17 alpha-hydroxyl group occurred earlier than that of 17-hydroxylated C21-steroids. Despite the decline in total corticosteroid production, the long term effect of ACTH was to enhance the relative secretion of 17 alpha-hydroxylated steroids and C19-steroids. Adrenocortical cells incubated for 72 h in the presence of ACTH continued to secrete cortisol, 17 alpha-hydroxyprogesterone, 11-deoxycortisol, and 11 beta-hydroxyandrostenedione in increased amounts. In fact, 11-deoxycortisol became a major secretory product of the ACTH-refractory adrenocortical cell. These results are indicative that ACTH acts in diverse manners on the bovine adrenocortical cell to affect corticosteroid secretion. The initial stimulation of corticosteroid production appears to be reflective of an increase in overall substrate (cholesterol) utilization and probably is mediated, in part, by an increase in cholesterol side chain cleavage activity. The secretion of 17 alpha-hydroxysteroids and C19-steroids is enhanced further by an action of ACTH to increase 17 alpha-hydroxylase activity and possibly also 17,20-lyase activity. The ACTH-induced refractoriness in corticosteroid production, on the other hand, appears to result primarily from a decline in precursor (cholesterol) utilization.
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13 MeSH Terms