Critical residues for the specificity of cofactors and substrates in humanestrogenic 17 beta-hydroxysteroid dehydrogenase 1: Variants designed from the three- dimensional structure of the enzyme

Human estrogenic 17 beta -hydroxysteroid dehydrogenase is an NADP(H)-prefer ring enzyme. It possesses 11- and 4-fold higher specificity toward NADP(H) over NAD(H) for oxidation and reduction, respectively, as demonstrated by k inetic studies. To elucidate the roles of the amino acids involved in cofac tor specificity, we generated variants by site-directed mutagenesis. The re sults showed that introducing a positively charged residue, lysine, at the Ser12 position increased the enzyme's preference for NADP(H) more than 20-f old. Substitution of the negatively charged residue, aspartic acid, into th e Leu36 position switched the enzyme's cofactor preference from NADPH to NA D with a 220-fold change in the ratio of the specificity toward the two cof actors in the case of oxidation. This variant dramatically abolished the en zyme's reductase function and stimulated its dehydrogenase activity, as sho wn by enzyme activity in intact cells. The substrate-binding pocket was als o studied with four variants: Ser142Gly, Ser142Cys, His221Ala, and Glu282Al a. The Ser142Gly variant abolished most of the enzyme's oxidation and reduc tion activities. The residual reductase activity in vitro is less than 2% t hat of the wild-type enzyme. However, the Ser142Cys variant was fully inact ive, both as a partially purified protein and in intact cells. This suggest s that the bulky sulfhydryl group of cysteine entirely disrupted the cataly tic triad and that the Ser142 side chain is important for maintaining the i ntegrity of this triad. His221 variation weakened the apparent affinity for estrone, as demonstrated by a 30-fold increase in Michaelis-Menten constan t, supporting its important role in substrate binding. This residue may pla y an important role in substrate inhibition via the formation of a dead-end complex. The formerly suggested importance of GIu282 could not be confirme d.