Characterization of the Substrate Binding Site in Rat Liver 3α-Hydroxysteroid/Dihydrodiol Dehydrogenase

Abstract

Rat liver 3-hydroxysteroid dehydrogenase (3-HSD), a member of the aldoketoreductase superfamily, inacti-vates circulating steroid hormones using NAD(P)H as cofactor. Despite determination of the 3-HSDNADP binary complex structure, the functional elements that dictate the binding of steroids remain unclear (Bennett, M.J., Schlegel, B.P., Jez, J.M., Penning, T.M., and Lewis, M. (1996) Biochemistry 35, 10702-10711). Two trypto-phans (Trp 86 and Trp 227) near the active site may have roles in substrate binding, and their fluorescence may be quenched upon binding of NADPH. Trp 86 is located within an apolar cleft, while Trp 227 is found on an opposing loop near the active site. A third tryptophan, Trp 148 , is on the periphery of the structure. To investigate the roles of these tryptophans in protein fluores-cence and ligand binding, we generated three mutant enzymes (W86Y, W148Y, and W227Y) by site-directed mu-tagenesis. Spectroscopic measurements on these proteins showed that Trp 148 contributed the most to the enzyme fluorescence spectra, with Trp 227 adding the least. Trp 86 was identified as the tryptophan quenched by bound NADPH through an energy transfer mechanism. The W86Y mutant altered binding of cofactor (a 3-fold increase in K d for NADPH) and steroid (a 7-fold increase in K d for testosterone). This mutation also dramatically decreased the catalytic efficiency observed with one-, two-, and three-ring substrates and decreased the binding affinity for nonsteroidal anti-inflammatory drugs but had little effect on the binding of aldose re-ductase inhibitors. Interestingly, mutation of Trp 227 significantly impaired steroid binding (a 22-fold increase in K d for testosterone), but did not alter binding of co-factor, smaller substrates, or inhibitors. Kinetically, the W148Y mutant was similar to wild-type enzyme. Our results demonstrate that Trp 86 and the apolar cleft is part of the substrate binding pocket. In addition, we propose a role for Trp 227 and its associated loop in binding steroids, but not small substrates or inhibitors, most likely through interaction with the C-and D-rings of the steroid. This work provides the first evidence that tryp-tophans on opposite sides of the apolar cleft are part of the steroid binding pocket and suggests how the enzyme may discriminate between nonsteroidal anti-inflamma-tory drugs and aldose reductase inhibitors like zopolres-tat. A model of how androstanedione binds in the apolar cleft is developed. These data provide further evidence that loop structures in members of the aldoketoreduc-tase superfamily are critical determinants of ligand binding.