ENZYMATIC AND NONENZYMATIC POLARIZATIONS OF ALPHA,BETA-UNSATURATED KETOSTEROIDS AND PHENOLIC STEROIDS - IMPLICATIONS FOR THE ROLES OF HYDROGEN-BONDING IN THE CATALYTIC MECHANISM OF DELTA(5)-3-KETOSTEROID ISOMERASE

Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 1 7 beta-estradiol and 17 beta-dihydroequilenin), which are potent compe titive inhibitors of Delta(5)-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-nortestostero ne, which occurs in the enzyme-steroid complex, resembles that observe d when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17 beta-estradiol and 17 beta-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate sp ecies in aqueous basic solutions. Since most enzymes bind their substr ates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environmen ts is unfavorable, we investigated the possibility that the spectral p erturbations 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 lambda( max) of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyand rost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, sh ows a lambda(max) of 258.5 nm in aqueous solution. This 17.5 nm differ ence in lambda(max) increases, as the solvent polarity is lowered, to a difference of 24.0 nm in hexane, presumably because there is less co mpetition for hydrogen bond formation by the less polar solvent molecu les. 2-Hydroxybenzoic acid showed progressively increasing red shifts and enhancements of UV absorption as the polarity of solvents was decr eased, and these changes resembled those of 17 beta-estradiol when bou nd to isomerase. The spectral changes of 17 beta-dihydroequilenin, whe n bound to isomerase, are better approximated by those of 1-acetyl-2-n aphthol in nonpolar solvents, which strengthen the intramolecular hydr ogen bond, than by ionization of 17 beta-dihydroequilenin in strong aq ueous base. Both fluorescence emission and excitation spectra of 17 be ta-dihydroequilenin in aqueous solution can be significantly altered b y high concentrations of hydrogen bond accepters such as malonate, and these changes closely mimic the spectral properties of 17 beta-dihydr oequilenin bound to isomerase. These results indicate that strong, dir ectional 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 i nteractions in the enzyme-inhibitor complexes implicate a low-barrier hydrogen bond between Tyr-14 and the enolic intermediate during cataly sis.