Irreversible active-site-directed inhibition of Δ5-3-ketosteroid isomerase by steroidal 17-β-oxiranes. Evidence for two modes of binding in steroid-enzyme complexes

Orientation, accessibility, and mobility of equilenin bound to the active site of steroid isomerase

“…The irreversibly bound form places the D ring of the steroid near Asp-38, whereas the initial reversibly bound complex has the A ring near Asp-38. The orientation with the aromatic A ring near the base of the hydrophobic binding pit (the same orientation proposed for the substrate in catalysis) is the predominant one (Bevins et al, 1980(Bevins et al, , 1986. This orientation shows UV spectral properties of an ionized phenol, as previously observed for dihydroequilenin (Wang et al, 1963) and estradiol (Wang et al, 1963;Kuliopulos et al, 1989).…”

“…Several lines of evidence suggest that steroids may bind to the isomerase in more than one orientation. We have shown that both 3/3-and 17/3-oxiranes inactive the isomerase in an active-site-directed process (Pollack et al, 1979;Bevins et al, 1980). Each forms a covalent bond to Asp-38 (Kayser et al, 1983;, a group known to be at the active site (Martyr & Benisek, 1975;Ogez et al, 1977),…”

“…showing that both ends of a steroid molecule can have access to the same part of the enzyme active site. The fact that only the /3-isomers inactivate the enzyme (Pollack et al, 1979;Bevins et al, 1980) is evidence for these two binding modes being related by a 180°rotation about an axis perpendicular to the plane of the steroid nucleus. Furthermore, the second-order rate constants for inactivation by a variety of oxiranes (both 3/3 and 17/3) are similar, consistent with a similar binding affinity for both modes (Bevins et al, 1986).…”

“…Investigations from one of our laboratories have shown that steroids can bind to the isomerase in two different orientations, related by a 180°rotation about an axis perpendicular to the plane of the steroid ring system (Bevins et al, 1980(Bevins et al, , 1986Kayser et al, 1983;Kashino et al, 1987;. Furthermore, it appears that for many steroids the binding constants are similar for the two modes of binding (Bevins et al, 1986).…”

Nature of the intermediate in the 3-oxo-.DELTA.5-steroid isomerase reaction

“…Alternatively, the lack of protonation of 4 at the active site of KSI might come from an incorrect orientation of the steroid molecule that does not allow interaction between Tyr-14 of KSI and the steroid. Nonproductive modes of binding have been observed for a variety of steroids with KSI (Bevins et al, 1980(Bevins et al, , 1986; Kayser et al, 1983;Bounds & Pollack, 1987). However, it is likely that at least a portion of the steroid is bound to KSI in an orientation that would allow interaction between Tyr-14 and the amine group of 4 but that proton transfer does not take place.…”