The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities

Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5α-androst-16-en-3-one (5α-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-β synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17α-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-β synthase as well as the 17α-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17α-hydroxylase (p < 0.013), a transient increase in C17,20 lyase, and an increase in andien-β synthase activity (p < 0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17α-hydroxylase, but did not affect the andien-β synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-β synthase activity of CYP17A1.     

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.     

Xenopus laevis CYP17 regulates androgen biosynthesis independent of the cofactor cytochrome b5

The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17alpha-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity). Most mammalian CYP17 isoforms have high 17alpha-hydroxylase relative to 17,20-lyase activities and preferentially mediate one of the two 17,20-lyase reactions. In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary. CYP17 isoforms generally rely on the cofactor cytochrome b(5) for the 17,20-lyase reaction, suggesting that the high lyase activity of Xenopus CYP17 might be due to a lesser dependence on b(5). The kinetics of Xenopus CYP17 expressed in yeast microsomes were therefore examined in the absence and presence of Xenopus on human b(5). Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b(5), confirming that the activity did not require b(5). However, both Xenopus and human b(5) slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b(5). Surprisingly, only the human b(5) cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17. Studies using human/Xenopus chimeric b(5) proteins revealed that human b(5) residues 16-41 were important for the specific regulation of the lyase activity of HuCYP17, possibly serving as an interacting domain with the enzyme. CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.     

Cytochrome b5 augments 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase activity

During adrenal steroidogenesis the competition between 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD) and cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1) for Δ(5) steroid intermediates greatly influences steroidogenic output. Cytochrome-b(5) (Cyt-b(5)), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b(5) on 3βHSD activity was investigated. In COS-1 cells, Cyt-b(5) was shown to significantly increase the activity of both caprine and ovine 3βHSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b(5) to have no influence on the K(m) values while significantly increasing the V(max) values of ovine 3βHSD for all its respective substrates. In addition, the activity of ovine 3βHSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b(5) or anti-Cyt-b(5) IgG. The results presented in this study indicate that Cyt-b(5) augments 3βHSD activity and represents the first documentation of such augmentation in any species.     

CYP17 mutation E305G causes isolated 17,20-lyase deficiency by selectively altering substrate binding

Cytochrome p450c17 (CYP17) converts the C21 steroids pregnenolone and progesterone to the C19 androgen precursors dehydroepiandrosterone (DHEA) and androstenedione, respectively, via sequential 17alpha-hydroxylase and 17,20-lyase reactions. Disabling mutations in CYP17 cause combined 17alpha-hydroxylase/17,20-lyase deficiency, but rare missense mutations cause isolated loss of 17,20-lyase activity by disrupting interactions of redox partner proteins with CYP17. We studied an adolescent male with clinical and biochemical features of isolated 17,20-lyase deficiency, including micropenis, hypospadias, and gynecomastia, who is homozygous for CYP17 mutation E305G, which lies in the active site. When expressed in HEK-293 cells or Saccharomyces cerevisiae, mutation E305G retains 17alpha-hydroxylase activities, converting pregnenolone and progesterone to 17alpha-hydroxysteroids. However, mutation E305G lacks 17,20-lyase activity for the conversion of 17alpha-hydroxypregnenolone to DHEA, which is the dominant pathway to C19 steroids catalyzed by human CYP17 (the delta5-steroid pathway). In contrast, mutation E305G exhibits 11-fold greater catalytic efficiency (kcat/Km) for the cleavage of 17alpha-hydroxyprogesterone to androstenedione compared with wild-type CYP17. We conclude that mutation E305G selectively impairs 17,20-lyase activity for DHEA synthesis despite an increased capacity to form androstenedione. Mutation E305G provides genetic evidence that androstenedione formation from 17alpha-hydroxyprogesterone via the minor delta4-steroid pathway alone is not sufficient for complete formation of the male phenotype in humans.     

Assessment of the ability of type 2 cytochrome b5 to modulate 17,20-lyase activity of human P450c17

The 17 alpha-hydroxylase and 17,20-lyase activities of P450c17 lead to the production of 17 alpha-hydroxypregnenolone (17 alpha-OH-Preg) and dehydroepiandrosterone (DHEA), respectively, in different tissues. The mechanisms of differential regulation of these two activities are not yet fully elucidated. It has been previously shown that cytochrome b5 (cyt-b5) could facilitate the 17,20-lyase activity of human P450c17. Recently, a cDNA (type 2 cyt-b5) sharing 45.8% homology with type 1 cyt-b5 has been isolated from human testis. Since high 17,20-lyase activity is required for the production of androgens in the testis, we wanted to determine the importance of this second cDNA in the modulation of P450c17 17,20-lyase activity and hence, its role in the formation of active androgens. We therefore isolated type 2 cyt-b5 from human testis by RT-PCR and analyzed, by transient transfection in transformed human embryonic kidney cells (HEK-293) of various amounts of vectors expressing cyt-b5, P450-reductase and P450c17, its ability to modulate the 17,20-lyase activity of human P450c17. Results show that, in the presence of NADPH cytochrome P450 reductase (P450-red), type 2 cyt-b5 increases 17,20-lyase activity to a level comparable to that of type 1. These results support the idea that types 1 and 2 cyt-b5 could be involved in the differential modulation of 17 alpha-hydroxylase and 17,20-lyase activities of P450c17. Furthermore, the analysis of mRNA expression of types 1 and 2 cyt-b5 by RT-PCR using primers specific to each type showed that both types are present in the liver but also in the adrenal and testis.     

Baboon cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17).

Human cytochrome P450 17alpha-hydroxylase (CYP17) catalyses not only the 17alpha-hydroxlation of pregnenolone and progesterone and the C17,20-side chain cleavage (lyase) of 17alpha-hydroxypregnenolone, necessary for the biosynthesis of C21-glucocorticoids and C19-androgens, but also catalyses the 16alpha-hydroxylation of progesterone. In efforts to understand the complex enzymology of CYP17, structure/function relationships have been reported previously after expressing recombinant DNAs, encoding CYP17 from various species, in nonsteroidogenic mammalian or yeast cells. A major difference between species resides in the lyase activity towards the hydroxylated intermediates and in the fact that the secretion of C19-steroids take place, in some species, principally in the gonads. Because human and higher primate adrenals secrete steroids, CYP17 has been characterized in the Cape baboon, a species more closely related to humans, in an effort to gain a further understanding of the reactions catalysed by CYP17. Baboon and human CYP17 cDNA share 96% homology. Baboon CYP17 has apparent Km and V values for pregnenolone and progesterone of 0.9 micro m and 0.4 nmol.h-1.mg protein-1 and 6.5 micro m and 3.9 nmol.h-1.mg protein-1, respectively. Baboon CYP17 had a significantly higher activity for progesterone hydroxylation relative to pregnenolone. No 16alpha-hydroxylase and no lyase activity for 17alpha-hydroxyprogesterone. Sequence analyses showed that there are 28 different amino acid residues between human and baboon CYP17, primarily in helices F and G and the F-G loop.     

Phosphorylation of Human Cytochrome P450c17 by p38α Selectively Increases 17,20 Lyase Activity and Androgen Biosynthesis

Cytochrome P450c17, a steroidogenic enzyme encoded by the CYP17A1 gene, catalyzes the steroid 17α-hydroxylation needed for glucocorticoid synthesis, which may or may not be followed by 17,20 lyase activity needed for sex steroid synthesis. Whether or not P450c17 catalyzes 17,20 lyase activity is determined by three post-translational mechanisms influencing availability of reducing equivalents donated by P450 oxidoreductase (POR). These are increased amounts of POR, the allosteric action of cytochrome b₅ to promote POR-P450c17 interaction, and Ser/Thr phosphorylation of P450c17, which also appears to promote POR-P450c17 interaction. The kinase(s) that phosphorylates P450c17 is unknown. In a series of kinase inhibition experiments, the pyridinyl imidazole drugs SB202190 and SB203580 inhibited 17,20 lyase but not 17α-hydroxylase activity in human adrenocortical HCI-H295A cells, suggesting an action on p38α or p38β. Co-transfection of non-steroidogenic COS-1 cells with P450c17 and p38 expression vectors showed that p38α, but not p38β, conferred 17,20 lyase activity on P450c17. Antiserum to P450c17 co-immunoprecipitated P450c17 and both p38 isoforms; however, knockdown of p38α, but not knockdown of p38β, inhibited 17,20 lyase activity in NCI-H295A cells. Bacterially expressed human P450c17 was phosphorylated by p38α in vitro at a non-canonical site, conferring increased 17,20 lyase activity. This phosphorylation increased the maximum velocity, but not the Michaelis constant, of the 17,20 lyase reaction. p38α phosphorylates P450c17 in a fashion that confers increased 17,20 lyase activity, implying that the production of adrenal androgens (adrenarche) is a regulated event.     

Active site proton delivery and the lyase activity of human CYP17A1

Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.     

Cytochrome P-450(17 alpha,lyase)-mediating pathway of androgen synthesis in bovine adrenocortical cultured cells.

Cytochrome P-450(17 alpha,lyase) mediating pathway of dehydroepiandrosterone (DHA) formation from pregnenolone was investigated in primary cultures of bovine adrenocortical fasciculata-reticularis cells. To determine whether DHA formation proceeds predominantly by successive monooxygenase reactions without 17 alpha-hydroxypregnenolone leaving P-450(17 alpha,lyase) the cells were incubated with [14C]pregnenolone and 17 alpha-[3H]hydroxypregnenolone in the presence of Trilostane. Results of the double-substrate double-label experiments indicate that in the presence of high concentration of pregnenolone most of DHA was formed, directly from pregnenolone by the successive reactions. Since the concentration of pregnenolone usually exceeds that of 17 alpha-hydroxypregnenolone in the adrenal glands, DHA is concluded to be formed predominantly by successive reactions from pregnenolone without 17 alpha-hydroxypregnenolone leaving P-450(17 alpha,lyase) in vivo. By chronic ACTH treatment, the activities of 17 alpha-hydroxylation and DHA formation in adrenocortical cultured cells became higher concomitantly with the increase of P-450(17 alpha,lyase) content. Most of DHA was found to be formed by successive reactions from pregnenolone even under such conditions.     

Homozygous CYP17A1 mutation (H373L) identified in a 46,XX female with combined 17α-hydroxylase/17,20-lyase deficiency

Background: Defects in cytochrome P450c17 are uncommon forms of congenital adrenal hyperplasia caused by CYP17A1 mutations. An H373L mutation in the CYP17A1 gene has been identified in Japanese and Chinese patients. This mutation impairs 17α-hydroxylase and 17,20-lyase activity. Case: A 23-year-old Korean female (46,XX) presented with absent spontaneous puberty and hypertension. Hormonal findings were consistent with combined 17α-hydroxylase/17,20-lyase deficiency. Very high levels of progesterone and 11-deoxycorticosterone were detected, coincident with normal 17-hydroxysteroid levels. Plasma levels of dehydroepiandrosterone, androstenedione and testosterone were extremely low. Mutation analysis of the CYP17A1 gene identified a homozygous missense mutation changing His (CAC) to Leu (CTC) at codon 373. This mutation is known to completely abolish both 17α-hydroxylase and 17,20-lyase activity. The patient's nonconsanguineous parents were heterozygous for this mutation. Of note, her serum steroid levels indicated decreased, but still present, 17α-hydroxylase activity in vivo. Conclusion: We detected a homozygous H373L mutation in a patient with combined 17α-hydroxylase/17,20-lyase deficiency. Our findings demonstrate minimally preserved 17α-hydroxylase activity in vivo and contribute to our knowledge of the regional prevalence of this mutation in Northeast Asia.     

Missense mutation serine106----proline causes 17 alpha-hydroxylase deficiency.

Steroid 17 alpha-hydroxylase deficiency is caused by defects in cytochrome P450c17, the single enzyme that has 17-alpha hydroxylase and 17,20-lyase activities. We describe a rapid and efficient polymerase chain reaction tactic for identifying these genetic lesions and identify Ser106----Pro as the cause of 17 alpha-hydroxylase deficiency in two unrelated homozygous patients from Guam. We used site-directed mutagenesis of the normal P450c17 cDNA to construct the Pro106 mutant, and expressed both the normal and mutant sequences in monkey COS-1 cells and in yeast. Expression of the normal sequence permitted the cells to convert pregnenolone to 17-OH pregnenolone, progesterone to 17-OH progesterone, and 17-OH pregnenolone to dehydroepiandrosterone, showing the normal sequence conferred both 17 alpha-hydroxylase and 17,20-lyase activities. Expression of the mutant sequence generated P450c17 mRNA, but conferred none of these activities, proving that the Ser106----Pro mutation abolished the 17 alpha-hydroxylase and 17,20-lyase activities. An HhaI restriction site created by the mutation should permit screening of large populations.     

Molecular basis of apparent isolated 17,20-lyase deficiency: compound heterozygous mutations in the C-terminal region (Arg(496)----Cys, Gln(461)----Stop) actually cause combined 17 alpha-hydroxylase/17,20-lyase deficiency.

The molecular defect in a reported case of isolated 17,20-lyase deficiency in a 46XY individual has been elucidated. The patient was found to be a compound heterozygote, carrying two different mutant alleles in the CYP17 gene. One allele contains a point mutation of arginine (CGC) to cysteine (TGC) at amino acid 496 in exon 8. The second allele contains a stop codon (TAG) in place of glutamine (CAG) at position 461 in exon 8 which is located 19 amino acids to the carboxy-terminal side of the P-450(17) alpha heme binding cysteine. COS-1 cells transfected with cDNAs containing one or the other of these mutations showed dramatically reduced 17 alpha-hydroxylase and 17,20-lyase activities relative to cells transfected with the wild type P-450(17) alpha cDNA. While the in vitro data in COS 1 cells can explain the patient's physical phenotype, with female external genitalia, it was somewhat discordant with the clinical expression of isolated 17,20-lyase deficiency with relative preservation of 17 alpha-hydroxylase activity in vivo. In addition to the expression studies of these two examples of mutants in the C-terminal region of cytochrome P-450(17) alpha, a third mutant cDNA construct containing a 4-base duplication at codon 480 previously found in patients with combined 17 alpha-hydroxylase/17,20-lyase deficiency was also expressed in COS-1 cells. This expressed protein was completely inactive with respect to both activities, supporting the biochemical findings in serum and in vitro biochemical data obtained using a testis from the patient. The results from these patients clearly indicate the importance of the C-terminal region of human P-450(17) alpha in its enzymatic activities.     

The submicrosomal distribution in rat and boar testis of some enzymes involved in androgen and 16-androstene biosynthesis

The submicrosomal localization of enzymes involved in androgen and 16-androstene biosynthesis in rat and boar testis tissue has been studied. 16-Androstene production was not evident in the rat testis but two enzymes concerned with androgen generation (17α-hydroxy C₂₁ steroid C-17,20 lyase; 17β-hydroxysteroid dehydrogenase, were found predominantly in agranular microsomes.In the boar testis the C-17,20 lyase had a similar distribution to the rat enzyme but the 17β-hydroxysteroid dehydrogenase was found be evenly distributed between the two microsomal types. The enzyme system, “andien-β synthetase”, involved in the conversion of pregnenolone to 5,16-androstadien-3β-ol, was found mainly (66%) in the agranular microsomes but had a lower specific activity than those of the two enzymes of androgen biosynthesis that have been studied.     

Side-chain cleavage of C21 steroids by testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/lyase): involvement of heme

The two steps in the side-chain cleavage of C21 steroids to give C19 steroids (i.e. 17 alpha-hydroxylation and C17,20 lyase activity) were examined using a highly purified cytochrome P-450 from microsomes of neonatal pig testis to determine the photochemical action spectra for the two reactions. Photochemical action spectra, using either 4-ene (progesterone) or 5-ene (pregnenolone) substrates, showed maximal reversal of inhibition by CO with light of 451 nm. Evidently the heme of cytochrome P-450 is involved in both 17 alpha-hydroxylation and in C17,20-lyase activity as in the case of the side-chain cleavage of cholesterol. Mechanisms proposed to account for enzymatic cleavage of the alpha-ketol side-chain of C21 steroids (C17,20 lyase activity) must be consistent with these findings.     

Role of AMP-activated protein kinase on steroid hormone biosynthesis in adrenal NCI-H295R cells

Regulation of human androgen biosynthesis is poorly understood. However, detailed knowledge is needed to eventually solve disorders with androgen dysbalance. We showed that starvation growth conditions shift steroidogenesis of human adrenal NCI-H295R cells towards androgen production attributable to decreased HSD3B2 expression and activity and increased CYP17A1 phosphorylation and 17,20-lyase activity. Generally, starvation induces stress and energy deprivation that need to be counteracted to maintain proper cell functions. AMP-activated protein kinase (AMPK) is a master energy sensor that regulates cellular energy balance. AMPK regulates steroidogenesis in the gonad. Therefore, we investigated whether AMPK is also a regulator of adrenal steroidogenesis. We hypothesized that starvation uses AMPK signaling to enhance androgen production in NCI-H295R cells. We found that AMPK subunits are expressed in NCI-H295 cells, normal adrenal tissue and human as well as pig ovary cells. Starvation growth conditions decreased phosphorylation, but not activity of AMPK in NCI-H295 cells. In contrast, the AMPK activator 5-aminoimidazole-4-carboxamide (AICAR) increased AMPKα phosphorylation and increased CYP17A1-17,20 lyase activity. Compound C (an AMPK inhibitor), directly inhibited CYP17A1 activities and can therefore not be used for AMPK signaling studies in steroidogenesis. HSD3B2 activity was neither altered by AICAR nor compound C. Starvation did not affect mitochondrial respiratory chain function in NCI-H295R cells suggesting that there is no indirect energy effect on AMPK through this avenue. In summary, starvation-mediated increase of androgen production in NCI-H295 cells does not seem to be mediated by AMPK signaling. But AMPK activation can enhance androgen production through a specific increase in CYP17A1-17,20 lyase activity.     

The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b5-dependent acyl-carbon cleavage activities

CYP17 (17alpha-hydroxylase-17,20-lyase; also P450c17 or P450(17alpha)) catalyses the17alpha-hydroxylation of progestogens and the subsequent acyl-carbon cleavage of the 17alpha-hydroxylated products (lyase activity) in the biosynthesis of androgens. The enzyme also catalyses another type of acyl-carbon cleavage (direct cleavage activity) in which the 17alpha-hydroxylation reaction is by-passed. Human CYP17 is heavily dependent on the presence of the membrane form of cytochrome b(5) for both its lyase and direct cleavage activities. In the present study it was found that substitution of human CYP17 amino acids, Arg(347), Arg(358) and Arg(449), with non-cationic residues, yielded variants that were impaired in the two acyl-carbon bond cleavage activities, quantitatively to the same extent and these were reduced to between 3 and 4% of the wild-type protein. When the arginines were replaced by lysines, the sensitivity to cytochrome b(5) was restored and the acyl-carbon cleavage activities were recovered. All of the human mutant CYP17 proteins displayed wild-type hydroxylase activity, in the absence of cytochrome b(5). The results suggest that the bifurcated cationic charges at Arg(347), Arg(358) and Arg(449) make important contributions to the formation of catalytically competent CYP17.cytochrome b(5) complex. The results support our original proposal that the main role of cytochrome b(5) is to promote protein conformational changes which allow the iron-peroxo anion to form a tetrahedral adduct that fragments to produce the acyl-carbon cleavage products.     

Synthesis and biochemical evaluation of a range of sulfonated derivatives of 4-hydroxybenzyl imidazole as highly potent inhibitors of rat testicular 17α-hydroxylase/17,20-lyase (P-45017α)

We report the synthesis and biochemical evaluation of a range of 4-sulfonated derivatives of 4-hydroxybenzyl imidazole which has been targetted against the two components of 17α-hydroxylase/17,20-lyase (P-450_17α), namely, 17α-hydroxylase (17α-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are highly potent inhibitors possessing excellent selectivity towards the lyase component.     

Synthesis and biochemical evaluation of a range of (4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole-based compounds as potent inhibitors of 17α-hydroxylase/17,20-lyase (P45017α) derived from rat testicular microsomes

We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of (4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole against the two components of 17α-hydroxylase/17,20-lyase (P450_17α), namely, 17α-hydroxylase (17α-OHase) and 17,20-lyase (lyase). The results show the compounds to be highly potent inhibitors with limited selectivity towards the lyase component [e.g., toluene-4-sulfonic acid 4-imidazol-1-ylmethyl-phenyl ester (4) possessed an IC₅₀ value of 40 nM against 17α-OHase and 30 nM against lyase].