Characterization of the substrate binding site in rat liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase. The roles of tryptophans in ligand binding and protein fluorescence.

The Journal of Biological Chemistry
J M JezT M Penning

Abstract

Rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), a member of the aldoketoreductase superfamily, inactivates circulating steroid hormones using NAD(P)H as cofactor. Despite determination of the 3alpha-HSD.NADP+ 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 tryptophans (Trp86 and Trp227) near the active site may have roles in substrate binding, and their fluorescence may be quenched upon binding of NADPH. Trp86 is located within an apolar cleft, while Trp227 is found on an opposing loop near the active site. A third tryptophan, Trp148, is on the periphery of the structure. To investigate the roles of these tryptophans in protein fluorescence and ligand binding, we generated three mutant enzymes (W86Y, W148Y, and W227Y) by site-directed mutagenesis. Spectroscopic measurements on these proteins showed that Trp148 contributed the most to the enzyme fluorescence spectra, with Trp227 adding the least. Trp86 was identified as the tryptophan quenched by bound NADPH through an energy transfer mechanism. The W86Y mutant altered binding of cofactor (a 3-fold inc...Continue Reading

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