Enzymic aromatization of deuterium labelled testosterone and androst-4-ene-3,17-dione

Holland, Herbert L.; Taylor, Gregg J.

doi:10.1139/v81-406

Cited by 18 publications

(7 citation statements)

References 27 publications

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“…In contrast, the non-competitive KIE for CYP3A4-catalyzed testosterone-6β-hydroxylation of an allylic C-H bond is only 2–3 (26). Holland and colleague have reported on the intermolecular kinetic isotope effect of the 19-methyl position on testosterone with aromatase (CYP19A1) with a k H / k D of 2.3 for 19-[ 2 H]-testosterone and 3.2 for 19,19,19-[ 2 H 3 ]-testosterone substrates (47). This wide range in observed KIE values suggests that, although all cytochrome P450 hydroxylation reactions might incorporate hydrogen atom abstraction in their catalytic cycles, the structural features of these transition states are considerably variable.…”

Section: Discussionmentioning

confidence: 99%

Minor Activities and Transition State Properties of the Human Steroid Hydroxylases Cytochromes P450c17 and P450c21, from Reactions Observed with Deuterium-Labeled Substrates

Yoshimoto

Zhou²,

Peng

et al. 2012

Biochemistry

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The steroid hydroxylases CYP17A1 (P450c17, 17-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) catalyze progesterone hydroxylation at one or more sites within a 2 Å radius. We probed their hydrogen atom abstraction mechanisms and regiochemical plasticity with deuterium-labeled substrates: 17-[2H]-pregnenolone; 17-[2H]-, 16α-[2H]-, 21,21,21-[2H3]-, and 21-[2H]-progesterone; and 21,21,21-[2H3]-17-hydroxyprogesterone. Product distribution and formation rates with recombinant human P450-oxidoreductase and wild-type human CYP17A1 or mutation A105L (reduced progesterone 16α-hydroxylation) and wild-type human CYP21A2 or mutation V359A (substantial progesterone 16α-hydroxylation) were used to calculate intramolecular and intermolecular kinetic isotope effects (KIEs). The intramolecular KIEs for CYP17A1 and mutation A105L were 4.1 and 3.8, respectively, at H-17 and 2.9 and 5.1, respectively, at H-16α. Mutation A105L 21-hydroxylates progesterone (5% of products), and wild-type CYP17A1 also catalyzes a trace of 21-hydroxylation, which increases with 16α-[2H]- and 17-[2H]-progesterone. The intramolecular KIEs with CYP21A2 mutation V359A and progesterone were 6.2 and 3.8 at H-21 and H-16α, respectively. Wild-type CYP21A2 also forms a trace of 16α-hydroxyprogesterone, which increased with 21,21,21-[2H3]-progesterone substrate. Competitive intermolecular KIEs paralleled the intramolecular KIE values, with DV values of 1.4–5.1 and DV/K values of 1.8–5.1 for these reactions. CYP17A1 and CYP21A2 mutation V359A both 16α-hydroxylate 16α-[2H]-progesterone with 33–44% deuterium retention, indicating stereochemical inversion. We conclude that human CYP17A1 has progesterone 21-hydroxylase activity and human CYP21A2 has progesterone 16α-hydroxylase activity, both of which are enhanced with deuterated substrates. The transition states for C-H bond cleavage in these hydroxylation reactions are either significantly non-linear and/or asymmetric, and C-H bond breakage is partially rate-limiting for all reactions.

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Section: Discussionmentioning

confidence: 99%

Minor Activities and Transition State Properties of the Human Steroid Hydroxylases Cytochromes P450c17 and P450c21, from Reactions Observed with Deuterium-Labeled Substrates

Yoshimoto

Zhou²,

Peng

et al. 2012

Biochemistry

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“…We decided to compare the kinetics of inactivation of aromatase by 5a with those of the parent unlabeled 5. It has been shown (Holland & Taylor, 1981) that a kinetic isotope effect of 3.2 accompanies aromatization of a 19,19,19-2H3-labeled steroid. A tritium isotope effect of the same magnitude was observed for aromatization of 19,19,19-3H3-labeled substrate (Miyairi 6 Fishman, 1983).…”

Section: Discussionmentioning

confidence: 99%

Tritium release from [19-3H]-19,19-difluoroandrost-4-ene-3,17-dione during inactivation of aromatase

Furth¹,

Robinson²

1989

Biochemistry

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Aromatase is a cytochrome P-450 enzyme involved in the conversion of androst-4-ene-3,17-dione to estrogen via sequential oxidations at the 19-methyl group. Previous studies from this laboratory showed that 19,19-difluoroandrost-4-ene-3,17-dione (5) is a mechanism-based inactivator of aromatase. The mechanism of inactivation was postulated to involve enzymic oxidation at, and hydrogen loss from, the 19-carbon. The deuteriated analogue 5b has now been synthesized and shown to inactivate aromatase at the same rate as the nondeuteriated parent (5). We conclude that C19-H bond cleavage is not the rate-limiting step in the overall inactivation process caused by 5. [19-3H]-19,19-Difluoroandrost-4-ene-3,17-dione (5b) with specific activity of 31 mCi/mmol was also synthesized to study the release of tritium into solution during the enzyme inactivation process. Incubation of [19-3H]19,19-difluoroandrost-4-ene-3,17-dione with human placental microsomal aromatase at differing protein concentrations resulted in time-dependent NADPH-dependent, and protein-dependent release of tritium. This tritium release is not observed in the presence of (19R)-10 beta-oxiranylestr-4-ene-3,17-dione, a powerful competitive inhibitor of aromatase. We conclude that aromatase attacks the 19-carbon of 19,19-difluoroandrost-4-ene-3,17-dione, as originally postulated.

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“…Compound 5 was synthesized in racemic form according to known procedures (14,15) and purified by f lash chromatography (R f ϭ 0.26, 90͞10 hexane͞ethyl acetate). Compound 5 could not be cleanly deconjugated to substrate 3, therefore 3 was prepared directly from 5,6,7,8-tetrahydro-2-naphthol (Aldrich) by Birch reduction (16) Kinetics. The reactions were done at 22°C in 100 mM Mops, pH 7.5, with 5% CH 3 CN as cosolvent in the presence or absence of antibody.…”

Section: Methodsmentioning

confidence: 99%

On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use an enamine mechanism

Chao

Hoffman

Wirsching

et al. 1997

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

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Reactive immunization has emerged as a new tool for the study of biological catalysis. A powerful application resulted in catalytic antibodies that use an enamine mechanism akin to that used by the class I aldolases. With regard to the evolution of enzyme mechanisms, we investigated the utility of an enamine pathway for the allylic rearrangement exemplified by ⌬ 5 -3-ketosteroid isomerase (KSI; EC 5.3.3.1). Our aldolase antibodies were found to catalyze the isomerization of both steroid model compounds and steroids. The kinetic and chemical studies showed that the antibodies afforded rate accelerations up to a factor of 10 4 by means of an enamine mechanism in which imine formation was the rate-determining step. In light of our observations and the enzyme studies by other workers, we suggest that an enamine pathway could have been an early, viable KSI mechanism. Although this pathway is amenable to optimization for increased catalytic power, it appears that certain factors precluded its evolution in known KSI enzymes.Many chemical reactions can proceed by alternative mechanisms. Hence, if more than one energetically accessible pathway is available for a particular transformation, it might be reasonable to expect that enzymes exist that operate via each distinct route. Yet with few exceptions, most notably the aldolases (1), Nature has apparently chosen to select and refine one approach for the conversion of a given substrate to product. One plausible explanation is that chemistry, and not specificity, has been the most challenging problem to solve during the course of enzyme evolution (2, 3). Once a clear chemical solution is found, subtle active-site modifications then occur that improve specificity and efficiency.The process of reactive immunization allows the evolution in real time of antibody catalysts with defined mechanisms (4, 5). Hence, it is possible to test whether a chemical pathway other than one found in Nature is feasible within a protein framework and how this new pathway compares with that optimized by natural selection. In fact, this approach was used to procure monoclonal antibodies (mAbs) that catalyze the aldol reaction using a lysine-mediated enamine mechanism central to the class I aldolase enzymes (5). The mechanism of these catalysts contrasts with the class II aldolases, which invoke metal ion-general base components.The ⌬ 5 -3-ketosteroid isomerase (KSI; EC 5.3.3

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Enzymic aromatization of deuterium labelled testosterone and androst-4-ene-3,17-dione

Cited by 18 publications

References 27 publications

Minor Activities and Transition State Properties of the Human Steroid Hydroxylases Cytochromes P450c17 and P450c21, from Reactions Observed with Deuterium-Labeled Substrates

Minor Activities and Transition State Properties of the Human Steroid Hydroxylases Cytochromes P450c17 and P450c21, from Reactions Observed with Deuterium-Labeled Substrates

Tritium release from [19-3H]-19,19-difluoroandrost-4-ene-3,17-dione during inactivation of aromatase

On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use an enamine mechanism

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