The reaction of the active-site-directed irreversible inhibitor (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2′-oxiran]-3-ol (5β) with 3-oxo-Δ5-steroid isomerase has been monitored by repetitive scanning ultraviolet spectroscopy of a solution of 5β plus isomerase against a blank containing only 5β. Upon initial mixing of 5β with the isomerase an absorbance maximum at ca. 250 nm appears. With time, this peak decreases and is replaced with a new peak near 280 nm. These results directly demonstrate the existence of a transient enzyme-steroid intermediate in the inactivation reaction. The ultraviolet spectrum suggests that the steroid in the transient complex resembles the ionized phenol, while the phenolic group in the irreversibly bound complex is un-ionized. These spectral studies support our previous proposal that there are two enzyme-steroid complexes that are related by a 180° rotation about an axis perpendicular to the plane of the steroid nucleus. This hypothesis offers an explanation for the reaction of 17β-oxiranes with the same residue (Asp-38) that is thought to be involved in the catalytic mechanism. Two new oxiranes, (17S)-spiro[estra-1,3,5(10)-triene-17,2′-oxiran]-3β-ol (6β) and (17S)-spiro[5α-androstane-17,2′-oxiran]-3-one (8β), were also found to be potent active-site-directed irreversible inhibitors of the isomerase (k3/KI, = 31 M-1 s-1 and 340 M-1 s-1, respectively). The relationship of these results to the nature of the active site of the isomerase is discussed.
|Original language||English (US)|
|Number of pages||6|
|State||Published - 1986|
ASJC Scopus subject areas