Kinetic Solvent Isotope Effect in Human P450 CYP17A1-Mediated Androgen Formation: Evidence for a Reactive Peroxoanion Intermediate

Gregory, Michael C.; Denisov, Ilia G.; Grinkova, Yelena V.; Khatri, Yogan; Sligar, Stephen G.

doi:10.1021/ja4086403

Cited by 74 publications

(143 citation statements)

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“…Running the normal P450 17A1 reaction (with NADPH-P450 reductase and cytochrome b 5 ) in 95% D 2 O showed no significant change in the rate of conversion of 17␣-hydroxyprogesterone to androstenedione and a small but statistically significant change in the rate of oxidation of 17␣-hydroxypregnenolone to DHEA, with an apparent isotope effect of 0.83 ( Fig. 17), which is much less than the effect (0.39) reported by Gregory et al (23). Interpretation of solvent kinetic deuterium isotope effects is complex (75), in that protonation and deprotonation can occur throughout the amino acid side chains of an enzyme, not only on an iron-oxygen complex.…”

Section: B Ring Hydroxylation Of 16␣17␣-dihydroxyprogesterone By P45mentioning

confidence: 62%

“…However, there was not enough purified material recovered to determine the (Fig. 3B) is a reported inverse kinetic solvent deuterium isotope effect (0.39) reported by Sligar and co-workers (23). If the mechanism in Fig.…”

Section: B Ring Hydroxylation Of 16␣17␣-dihydroxyprogesterone By P45mentioning

confidence: 71%

“…iodosylbenzene, a single oxygen atom donor, and H 2 O 2 . Finally, we measured kinetic solvent isotope effects for the reactions, in light of inconsistencies in the field (23,35). Our evidence now suggests that an FeO 3ϩ mechanism is likely, at least in part, for the 17␣,20-lyase reaction, and we also demonstrate the ability of the enzyme to catalyze additional 6␤-and 16-hydroxylation reactions.…”

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confidence: 72%

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Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Yoshimoto

Gonzalez

Auchus

et al. 2016

Journal of Biological Chemistry

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We propose three mechanisms that can involve the FeO 3؉ entity and that explain the 18 O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17␣-hydroxylated products to yield 16,17␣-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6␤-hydroxylation of 16␣,17␣-dihydroxyprogesterone and the oxidation of both 16␣,17␣-dihydroxyprogesterone and 16␣,17␣-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17␣,20-lyase reaction cannot be excluded. Cytochrome P450 (P450)3 enzymes catalyze oxidations of more chemicals than any other group of proteins (1). The list of reactions includes aliphatic and aromatic hydroxylations, heteroatom oxidations, epoxidations, and reactions involving both ring formation and cleavage (2-4). Many P450 reactions are important in the biosynthesis and degradation of steroids and sterols (4, 5), including several critical C-C bond cleavage reactions, i.e. those catalyzed by P450s 11A1, 17A1 (Fig. 1), 19A1, and 51A1 (6, 7).The mechanisms of the C-C cleavage reactions have been the subject of considerable interest and debate. One of the questions with P450s 17A1, 19A1, and 51A1 has been whether the active oxidant is a ferric peroxide (FeO 2 Ϫ ), which is an early intermediate following oxygen addition to the iron (Fig. 2, step 4) or the FeO 3ϩ species (Fig. 2, step 6), often referred to as compound I (4, 10, 11). With P450s 17A1 and 19A1, a variety of approaches has been applied, including theoretical calculations, biomimetic models, spectroscopy, substrate atom labeling, and kinetics (12-32).These C-C bond cleavage reactions are complex, and many of the results are ambiguous; also, a "mixed" mechanism would not be discerned in many of these experiments. One powerful approach originally used by Akhtar and co-workers (27-31) analyzes the actual reaction and can provide discrimination between the nucleophilic FeO 2 Ϫ and electrophilic FeO 3ϩ reactions (Fig. 2), based on the incorporation of 18 O label from O 2 into the carboxylic acid products (Fig. 3) (7). However, these experiments are complicated due to the ubiquitous presence of formic acid (P450 19A1 and 51A1 reactions) and acetic acid (P450 17A1) in laboratory settings. Thus, the data from such experiments are interpreted with the most confidence when the steroid substrates are labeled with 2 H or 13 C isotopes to facilitate analysis (15,33). Even then, the mass spectrometry results can be problematic, particularly if a shift of only one atomic mass unit is introduced and isotopologues derived from 18 O incorporation are not discriminated from molecules containing natural abundance 13 C atoms (33). The incorporation of one atom of 18 O label from O 2 into formic acid (F...

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Section: B Ring Hydroxylation Of 16␣17␣-dihydroxyprogesterone By P45mentioning

confidence: 62%

Section: B Ring Hydroxylation Of 16␣17␣-dihydroxyprogesterone By P45mentioning

confidence: 71%

mentioning

confidence: 72%

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Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Yoshimoto

Gonzalez

Auchus

et al. 2016

Journal of Biological Chemistry

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“…Although the observation of this single feature, appearing at 791 cm −1 , is obviously consistent with an intermediate with "peroxo-like" character, it must be noted that this vibrational frequency and 16 O/ 18 O isotopic shifts are close to what is expected for the ν(Fe = O) mode of a ferryl heme species; the frequencies of such ferryl species depend on the transaxial ligand and distal pocket interactions, with reported values ranging from 745 cm −1 up to about 800 cm −1 (34,36). However, the assignment of this feature to a ferryl species was ruled out by experiments conducted with scrambled oxygen, a (1:2:1) mixture of 16 Fig.…”

Section: Resultsmentioning

confidence: 78%

“…Thus, although the hydroxylation reactions producing 17-OH PREG and 17-OH PROG are commonly accepted to be mediated by P450 compound I, the mechanism of the lyase reaction, converting 17-OH PREG to DHEA, has been intensively debated, based on results of extensive structural and functional studies (5,9,14,17,18). One of the proposed schemes is illustrated with red arrows in Fig.…”

Section: Significancementioning

confidence: 99%

Unveiling the crucial intermediates in androgen production

Mak

Gregory

Denisov

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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189

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Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C 17 -C 20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C 20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon-carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences.T he excessive production of androgen, which effectively fuels the progression of cancer, especially prostate cancer, was first treated by surgical methods (1), whereas more modern approaches are focused on the discovery and development of pharmaceuticals that can selectively inhibit androgen synthesis (2, 3). A member of the cytochrome P450 superfamily (4, 5), cytochrome P450 17A1 (CYP17A1), occupies a central role in the biosynthesis of steroid hormones in humans. As was first reported by Nakajin and Hall (6) and Nakajin et al. (7) for CYP17 from pig testis, this enzyme catalyzes two fundamentally different types of chemical transformations (5-11), with the first being the efficient hydroxylation of both of its primary substrates, pregnenolone (PREG) and progesterone (PROG), to 17-hydroxypregnenolone (17-OH) PREG and 17-hydroxypregnenolone, respectively (Fig. 1). Importantly, 17-OH PREG is further processed in a second step in which CYP17A1 now catalyzes, not another hydroxylation reaction, but a complex 17,20 carbon-carbon (C-C) bond cleavage (lyase) reaction. This step converts 17-OH PREG to dehydroepiandrosterone (DHEA), a process that represents a critical branch point in human steroidogenesis by providing the essential precursor to androgens and various corticosteroids (5-9). Although a similar C-C bond cleavage of 17-OH PROG is also mediated by CYP17A1, it is of less importance in humans because its efficiency is only about 2% of the efficiency of the physiologically important lyase reaction involving 17-OH PREG (10). Recognizing the intensive efforts currently underway to design and test substances that selectively inhibit CYP17A1 (2, 3), there is a pressing need to enhance our understanding of the relevant reaction mechanisms, a task that typically entails identification of key reaction intermediates. Dir...

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Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by myxobacterial CYP260B1

et al. 2016

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Although the oxidation of aldehydes to carboxylic acids is mainly catalyzed by aldehyde dehydrogenases in nature, cytochromes P450 are also able to perform such reactions. In this study, we demonstrate the oxidation of cinnamaldehyde to cinnamic acid by the myxobacterial CYP260B1. Following our docking studies of the aldehyde, we generated T224A and T234A mutants of CYP260B1 by site-directed mutagenesis to disrupt the substrate positioning and proton delivery, respectively. Furthermore, we used the kinetic solvent isotope effect on the steady-state turnover of the substrate to investigate the reactive intermediate capable of performing the catalysis. Our results suggest that the aldehyde oxidation occurs via a nucleophilic attack of the ferric peroxoanion.

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Kinetic Solvent Isotope Effect in Human P450 CYP17A1-Mediated Androgen Formation: Evidence for a Reactive Peroxoanion Intermediate

Cited by 74 publications

References 21 publications

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Unveiling the crucial intermediates in androgen production

Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by myxobacterial CYP260B1

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