Enzymatic and nonenzymatic polarizations of αβ-unsaturated ketosteroids and phenolic steroids. Implications for the roles of hydrogen bonding in the catalytic mechanism of Δ5-3-ketosteroid isomerase

Qinjian Zhao, Albert S. Mildvan, Paul Talalay

Research output: Contribution to journalArticle

Abstract

Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17β-estradiol and 17β-dihydroequilenin), which are potent competitive inhibitors of Δ5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme - steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17β-estradiol and 17β-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a λmax of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a λmax of 258.5 nm in aqueous solution. This 17.5 nm difference in λmax increases, as the solvent polarity is lowered, to a difference of 24.0 nm in hexane, presumably because there is less competition for hydrogen bond formation by the less polar solvent molecules. 2-Hydroxybenzoic acid showed progressively increasing red shifts and enhancements of UV absorption as the polarity of solvents was decreased, and these changes resembled those of 17β-estradiol when bound to isomerase. The spectral changes of 17β-dihydroequilenin, when bound to isomerase, are better approximated by those of 1-acetyl-2-naphthol in nonpolar solvents, which strengthen the intramolecular hydrogen bond, than by ionization of 17β-dihydroequilenin in strong aqueous base. Both fluorescence emission and excitation spectra of 17β-dihydroequilenin in aqueous solution can be significantly altered by high concentrations of hydrogen bond acceptors such as malonate, and these changes closely mimic the spectral properties of 17β-dihydroequilenin bound to isomerase. These results indicate that strong, directional hydrogen bond(s) to the functional groups of steroids in the solvent-inaccessible active site can explain the spectral behavior of these steroids when bound to isomerase. Such strong hydrogen-bonding interactions in the enzyme - inhibitor complexes implicate a low-barrier hydrogen bond between Tyr-14 and the enolic intermediate during catalysis.

Original languageEnglish (US)
Pages (from-to)426-434
Number of pages9
JournalBiochemistry®
Volume34
Issue number2
StatePublished - 1995

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steroid delta-isomerase
Ketosteroids
Hydrogen Bonding
Hydrogen
Hydrogen bonds
Isomerases
Steroids
Polarization
Nandrolone
Estradiol
Enzymes
Catalytic Domain
Comamonas testosteroni
Fluorescence
Methyl Ethers
Salicylic Acid
Hexanes
Enzyme Inhibitors
Catalysis
Hydroxyl Radical

ASJC Scopus subject areas

  • Biochemistry

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Enzymatic and nonenzymatic polarizations of αβ-unsaturated ketosteroids and phenolic steroids. Implications for the roles of hydrogen bonding in the catalytic mechanism of Δ5-3-ketosteroid isomerase. / Zhao, Qinjian; Mildvan, Albert S.; Talalay, Paul.

In: Biochemistry®, Vol. 34, No. 2, 1995, p. 426-434.

Research output: Contribution to journalArticle

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abstract = "Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17β-estradiol and 17β-dihydroequilenin), which are potent competitive inhibitors of Δ5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme - steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17β-estradiol and 17β-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a λmax of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a λmax of 258.5 nm in aqueous solution. This 17.5 nm difference in λmax increases, as the solvent polarity is lowered, to a difference of 24.0 nm in hexane, presumably because there is less competition for hydrogen bond formation by the less polar solvent molecules. 2-Hydroxybenzoic acid showed progressively increasing red shifts and enhancements of UV absorption as the polarity of solvents was decreased, and these changes resembled those of 17β-estradiol when bound to isomerase. The spectral changes of 17β-dihydroequilenin, when bound to isomerase, are better approximated by those of 1-acetyl-2-naphthol in nonpolar solvents, which strengthen the intramolecular hydrogen bond, than by ionization of 17β-dihydroequilenin in strong aqueous base. Both fluorescence emission and excitation spectra of 17β-dihydroequilenin in aqueous solution can be significantly altered by high concentrations of hydrogen bond acceptors such as malonate, and these changes closely mimic the spectral properties of 17β-dihydroequilenin bound to isomerase. These results indicate that strong, directional hydrogen bond(s) to the functional groups of steroids in the solvent-inaccessible active site can explain the spectral behavior of these steroids when bound to isomerase. Such strong hydrogen-bonding interactions in the enzyme - inhibitor complexes implicate a low-barrier hydrogen bond between Tyr-14 and the enolic intermediate during catalysis.",
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T1 - Enzymatic and nonenzymatic polarizations of αβ-unsaturated ketosteroids and phenolic steroids. Implications for the roles of hydrogen bonding in the catalytic mechanism of Δ5-3-ketosteroid isomerase

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N2 - Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17β-estradiol and 17β-dihydroequilenin), which are potent competitive inhibitors of Δ5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme - steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17β-estradiol and 17β-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a λmax of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a λmax of 258.5 nm in aqueous solution. This 17.5 nm difference in λmax increases, as the solvent polarity is lowered, to a difference of 24.0 nm in hexane, presumably because there is less competition for hydrogen bond formation by the less polar solvent molecules. 2-Hydroxybenzoic acid showed progressively increasing red shifts and enhancements of UV absorption as the polarity of solvents was decreased, and these changes resembled those of 17β-estradiol when bound to isomerase. The spectral changes of 17β-dihydroequilenin, when bound to isomerase, are better approximated by those of 1-acetyl-2-naphthol in nonpolar solvents, which strengthen the intramolecular hydrogen bond, than by ionization of 17β-dihydroequilenin in strong aqueous base. Both fluorescence emission and excitation spectra of 17β-dihydroequilenin in aqueous solution can be significantly altered by high concentrations of hydrogen bond acceptors such as malonate, and these changes closely mimic the spectral properties of 17β-dihydroequilenin bound to isomerase. These results indicate that strong, directional hydrogen bond(s) to the functional groups of steroids in the solvent-inaccessible active site can explain the spectral behavior of these steroids when bound to isomerase. Such strong hydrogen-bonding interactions in the enzyme - inhibitor complexes implicate a low-barrier hydrogen bond between Tyr-14 and the enolic intermediate during catalysis.

AB - Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17β-estradiol and 17β-dihydroequilenin), which are potent competitive inhibitors of Δ5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme - steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17β-estradiol and 17β-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a λmax of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a λmax of 258.5 nm in aqueous solution. This 17.5 nm difference in λmax increases, as the solvent polarity is lowered, to a difference of 24.0 nm in hexane, presumably because there is less competition for hydrogen bond formation by the less polar solvent molecules. 2-Hydroxybenzoic acid showed progressively increasing red shifts and enhancements of UV absorption as the polarity of solvents was decreased, and these changes resembled those of 17β-estradiol when bound to isomerase. The spectral changes of 17β-dihydroequilenin, when bound to isomerase, are better approximated by those of 1-acetyl-2-naphthol in nonpolar solvents, which strengthen the intramolecular hydrogen bond, than by ionization of 17β-dihydroequilenin in strong aqueous base. Both fluorescence emission and excitation spectra of 17β-dihydroequilenin in aqueous solution can be significantly altered by high concentrations of hydrogen bond acceptors such as malonate, and these changes closely mimic the spectral properties of 17β-dihydroequilenin bound to isomerase. These results indicate that strong, directional hydrogen bond(s) to the functional groups of steroids in the solvent-inaccessible active site can explain the spectral behavior of these steroids when bound to isomerase. Such strong hydrogen-bonding interactions in the enzyme - inhibitor complexes implicate a low-barrier hydrogen bond between Tyr-14 and the enolic intermediate during catalysis.

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