Structural determinants of catalysis and steroid binding in 3-alpha-hydroxysteroid dehydrogenase

The NAD(P)(H)-linked oxidoreductase rat liver 3$\alpha$-hydroxysteroid dehydrogenase (3$\alpha$-HSD, EC 1.1.1.213, AKR1C9) is a member of the aldo-keto reductase superfamily (AKR). The focus of this thesis was to identify the structural determinants of 3$\alpha$-HSD catalysis and steroid binding as a guide to inhibitor design. Crystallographic studies of 3$\alpha$-HSD have led to the proposal of a catalytic mechanism in which Y55 acts as a general acid and its pK value may be lowered by K84 and D50. Characterization of site-directed mutants suggest that Y55 makes the largest contribution to the chemical mechanism for steroid oxidoreduction, whereas the major contribution of K84 and H117 is to steroid binding. Surprisingly, the Y55 mutants were efficient catalysts of 9,10-phenanthrenequinone reduction. This is the first report whereby the invariant catalytic tyrosine in the AKRs has been mutated with retention of robust enzyme activity. The unique substrate specificity of Y55 mutants for polycyclic aromatic hydrocarbon o-quinones is reminiscent of $\zeta$-crystallin, an alcohol dehydrogenase (ADH) which lacks a catalytic zinc atom. This suggests a common functional role for tyrosine and zinc, possibly through the stabilization of an oxyanion transition state. A comparison of the active site residues from 3$\alpha$-HSD, ADH, 3$\alpha$,20$\beta$-HSD, lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase showed that (1) the C4 of the nicotinamide ring, (2) the tyrosine oxygen/zinc atom and (3) a histidine/threonine/serine side-chain are positionally conserved, despite a lack of sequence identity. These comparisons illustrate active site convergence in five structurally distinct classes of dehydrogenases. To identify regions of the enzyme involved in steroid hormone recognition, I employed mechanism-based inactivators. Secosteroids which contain latent Michael acceptors ($\alpha,\beta$-unsaturated alcohols) at opposite ends of the steroid nucleus inactivated 3$\alpha$-HSD only in the presence of NAD$\sp+$. The chemically prepared products of secosteroid oxidation ($\alpha,\beta$-unsaturated carbonyls) behaved as stoichiometric inactivators. These results demonstrated backwards binding of secosteroid substrates, and their turnover to reactive acetylenic ketones which alkylate 3$\alpha$-HSD. A C217A mutant was resistant to inactivation and supports a role for Cys-217 in inactivation by secosteroids. Our ability to model only the active secosteroid alcohol diastereomers into the active site illustrates the importance of steric fit in substrate specificity