Affinity alkylation of human placental 3β -hydroxy-5-ENE-steroid dehydrogenase and steroid 5→4-ene-isomerase by 2α-bromoacetoxyprogesterone: Evidence for separate dehydrogenase and isomerase sites on one protein

Thomas, James L.; Myers, Richard P.; Rosik, Leonard O.; Strickler, Ronaldo C.

doi:10.1016/0022-4731(90)90121-8

Cited by 23 publications

(12 citation statements)

References 26 publications

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“…17β-Acetoxy-trilostane was synthesized by reacting trilostane with acetic anhydride using pyridine as a catalyst in ethanol as previously described (Thomas et al, 1990). Silica gel thin-layer chromatography (benzene-ethyl acetate, 92:8, v/v) gave a single spot (R f = 0.33) that migrated farther than the starting steroid, trilostane (R f = 0.09).…”

Section: Methodsmentioning

confidence: 99%

Structural basis for the selective inhibition of human 3β-hydroxysteroid dehydrogenase 1 in human breast tumor MCF-7 cells

Thomas

Bucholtz

Sun

et al. 2009

Molecular and Cellular Endocrinology

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Human 3β-hydroxysteroid dehydrogenase/isomerase type 1 (3β-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a target enzyme for inhibition in the treatment of breast cancer in postmenopausal women. Human 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland in this population. In our recombinant human breast tumor MCF-7 Tet-off cells that express either 3β-HSD1 or 3β-HSD2, trilostane and epostane inhibit the DHEA-induced proliferation of MCF-7 3β-HSD1 cells with 12-to 16-fold lower IC 50 values compared to the MCF-7 3β-HSD2 cells. The compounds also competitively inhibit purified human 3β-HSD1 with 12-to 16-fold lower K i values compared to the noncompetitive Ki values measured for human 3β-HSD2. Using our structural model of 3β-HSD1, trilostane or 17β-acetoxytrilostane was docked in the active site of 3β-HSD1, and Arg195 in 3β-HSD1 or Pro195 in 3β-HSD2 was identified as a potentially critical residue (one of 23 nonidentical residues in the two isoenzymes). The P195R mutant of 3β-HSD2 were created, expressed and purified. Kinetic analyses of enzyme inhibition suggest that the high-affinity, competitive inhibition of 3β-HSD1 by trilostane and epostane may be related to the presence of Arg195 in 3β-HSD1 vs Pro195 in 3β-HSD2.

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Section: Methodsmentioning

confidence: 99%

Structural basis for the selective inhibition of human 3β-hydroxysteroid dehydrogenase 1 in human breast tumor MCF-7 cells

Thomas

Bucholtz

Sun

et al. 2009

Molecular and Cellular Endocrinology

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“…Assays that monitored the loss of 3β-HSD or isomerase activity during enzyme inactivation were performed in duplicate according to our published conditions (Thomas et al, 1990). The slope of the initial linear increase in absorbance at 340 nm (due to NADH production) per unit time was used to determine 3β-HSD activity.…”

Section: Methodsmentioning

confidence: 99%

“…Isomerase activity was measured by the initial absorbance increase at 241 nm (due to androstenedione formation from 5-androstene-3,17-dione) as a function of time. Blank assays (zeroenzyme, zero-substrate) assured that specific isomerase activity was measured as opposed to nonenzymic "spontaneous" isomerization (Thomas et al, 1990). Changes in absorbance were measured with a Varian (Palo Alto, CA) Cary 219 recording spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

Affinity Radiolabeling Identifies Peptides Associated with the Isomerase Activity of Human Type I (Placental) 3β-Hydroxysteroid Dehydrogenase/Isomerase

1997

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3beta-Hydroxysteroid dehydrogenase and steroid Delta5-->4-isomerase (3beta-HSD/isomerase) were purified as a single protein from human term placenta. The affinity alkylator, 5,10-secoestr-4-yne-3,10, 17-trione (secosteroid), was incubated with the purified enzyme (30/1 secosteroid/enzyme molar ratio) to produce an 80% loss of initial isomerase activity over 90 min in a time-dependent, irreversible manner. The secosteroid inactivated 3beta-HSD by only 20% during the same 90 min. Incubations containing the isomerase substrate steroid, 5-androstene-3,17-dione, completely protected the isomerase activity from inactivation by the secosteroid and did not slow the inactivation of 3beta-HSD. The enzyme containing covalently bound steroid was separated from unreacted secosteroid by reversed phase HPLC. Ketones on the protein-bound secosteroid were radiolabeled by reduction with sodium boro[3H]hydride (specific radioactivity 50 microCi/micromol for the transferred tritium). After removal of the unreacted sodium boro[3H]hydride, the affinity-radiolabeled enzyme was digested with trypsin-TPCK, and the peptides were isolated by reversed phase HPLC. The radiolabeled peptide fractions were sequenced. The secosteroid alkylated three tryptic peptides: 251GQFYYISDDTPHQSYDNLNYTLSK274, tritiated His262; 176NGGTLYTCALR186, tritiated Cys183; and 353TVEWVGSLVDR363, tritiated Trp356. Coincubation with the isomerase substrate blocked the labeling of these three peptides and shifted the alkylation by secosteroid to a single tryptic peptide (135EIIQNGHEEEPLENTWPAPYPHSK159, tritiated His142). Using substrate protection to validate specificity, the affinity labeling secosteroid has identified peptides in the enzyme that are associated with isomerase activity.

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“…In addition to the NADH/NAD ϩ protection (5,8) and the secosteroid inactivation (8) studies discussed above, NADH (20) and pregnenolone (19) protected the same tryptic peptides (Arg-250, Lys-175) in the enzyme from affinity radioalkylation by 2␣-bromo[2Ј- …”

Section: Discussionmentioning

confidence: 99%

“…Assays that monitored the loss of 3␤-HSD or isomerase activity during enzyme inactivation were performed on aliquots taken from the incubation mixtures at appropriate time intervals in duplicate according to our published conditions (5). The slope of the initial linear increase in absorbance at 340 nm (due to NADH production during the oxidation of pregnenolone) per unit time was used to determine 3␤-HSD activity.…”

Section: Synthesis and Stability Of 8-bdb-tadp-mentioning

confidence: 99%

An NADH-induced Conformational Change That Mediates the Sequential 3β-Hydroxysteroid Dehydrogenase/Isomerase Activities Is Supported by Affinity Labeling and the Time-dependent Activation of Isomerase

Thomas¹,

Frieden

Nash³

et al. 1995

Journal of Biological Chemistry

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3␤-Hydroxysteroid dehydrogenase (3␤-HSD, t 1/2 ‫؍‬ 85 s). A time lag is also observed for the activation of isomerase by NADH. This combination of affinity labeling and biophysical data using nucleotide derivatives supports our model for the sequential reaction mechanism; the cofactor product of the 3␤-HSD reaction, NADH, activates isomerase by inducing a conformational change in the single, bifunctional enzyme protein. 3␤-Hydroxy-⌬5 -steroid dehydrogenase (3␤-HSD, 1 EC 1.1.1.145) and steroid ⌬-isomerase (EC 5.3.3.1) sequentially catalyze the two step-conversion of 3␤-hydroxy-5-ene steroids to 3-keto-4-ene steroids on a single enzyme protein purified from human placenta (1), bovine adrenals (2), rat adrenals (3), or rat testis (4). With pregnenolone shown as a representative 3␤-hydroxy-5-ene substrate, the enzyme reactions are as follows:Pregnenolone 3 5-pregnene-3,20-dione 3 progesterone 3␤-HSD isomerase REACTION 1This reaction scheme shows the reduction of NAD ϩ to NADH by the 3␤-HSD activity and the requirement of coenzyme (without further conversion) for the activation of isomerase (1). Because 3␤-HSD and isomerase were inactivated at different rates by 2␣-bromoacetoxyprogesterone (5) and exhibited different types of inhibition by N, N-diethyl-4-methyl-3-oxo-4-aza-5␣-androstene-17␤-carboxamide (6), it appeared that the two activities were catalyzed at separate sites on the same enzyme.Our studies of purified human placental 3␤-HSD/isomerase have suggested that NADH mediates a shift in enzyme activity from dehydrogenase to isomerase by inducing a conformational change in the enzyme protein. NADH is the coenzyme product of the 3␤-HSD reaction and is a potent, essential activator of isomerase activity (7). NADH, but not NAD ϩ , completely protects both the 3␤-HSD and isomerase activities from inactivation by the substrate-site affinity alkylators, 2␣-bromoacetoxyprogesterone (5) and 5,10-secoestr-4-yne-3,10,17-trione (8). Finally, the isomerase-site-directed secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivated the isomerase activity with the expected first-order kinetics but inactivated the 3␤-HSD activity in an unexpected manner. As the concentration of the alkylating secosteroid increased, the rate of 3␤-HSD inactivation paradoxically decreased (8) instead of increasing in accordance with the Kitz and Wilson model of irreversible enzyme inhibition (9).In this report, the interaction of 3␤-HSD/isomerase with coenzyme is studied using the cofactor-site affinity alkylator, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5Ј-diphosphate (8-BDB-TADP). In addition, the time-dependent effects of NADH binding on the intrinsic enzyme fluorescence and on isomerase activation are measured. 8-BDB-TADP, as well as the 6-and 2-(4-bromo-2,3-dioxobutyl)thio-derivatives of ADP, have been

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Affinity alkylation of human placental 3β -hydroxy-5-ENE-steroid dehydrogenase and steroid 5→4-ene-isomerase by 2α-bromoacetoxyprogesterone: Evidence for separate dehydrogenase and isomerase sites on one protein

Cited by 23 publications

References 26 publications

Structural basis for the selective inhibition of human 3β-hydroxysteroid dehydrogenase 1 in human breast tumor MCF-7 cells

Structural basis for the selective inhibition of human 3β-hydroxysteroid dehydrogenase 1 in human breast tumor MCF-7 cells

Affinity Radiolabeling Identifies Peptides Associated with the Isomerase Activity of Human Type I (Placental) 3β-Hydroxysteroid Dehydrogenase/Isomerase

An NADH-induced Conformational Change That Mediates the Sequential 3β-Hydroxysteroid Dehydrogenase/Isomerase Activities Is Supported by Affinity Labeling and the Time-dependent Activation of Isomerase

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