Enzymic and Nonenzymic Polarizations of α, β-Unsaturated Ketosteroids and Phenolic Steroids. Implications for the Roles of Hydrogen Bonding in the Catalytic Mechanism of Δ⁵-3-Ketosteroid Isomerase

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 enzymesteroid 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 enzymebound 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 γ_maxof 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 γ_maxof 258.5 nm in aqueous solution. This 17.5 nm difference in Amax 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 17/S-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.