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., 17 beta-estradiol and 17 beta-dihydroequilenin), which are potent competitive 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-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 17 beta-estradiol and 17 beta-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 lambda max 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 lambda max of 258.5 nm in aqueous solution.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhao
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Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Mildvan
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AS
Talalay
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D000735:Androstenedione / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004855:Equilenin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007664:Ketosteroids / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010636:Phenols / Q000737:chemistry*; D011375:Progesterone Reductase / Q000737:chemistry* / Q000378:metabolism; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013251:Steroid Isomerases / Q000737:chemistry* / Q000378:metabolism
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D007664:Ketosteroids; D009097:Multienzyme Complexes; D010636:Phenols; D012997:Solvents; C037874:17-dihydroequilenin; C060445:4-methoxyandrostenedione; D000735:Androstenedione; D011375:Progesterone Reductase; D013251:Steroid Isomerases; C014594:formestane; D004855:Equilenin