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Characterization of Structural and Functional Properties of Human 17ß-Hydroxysteroid Dehydrogenase Type 1 Using Recombinant Enzymes and Site-Directed Mutagenesis

Biocenter Oulu and Department of Clinical Chemistry (T.P., M.P., P.V., R.V.) University of Oulu FIN-90220 Oulu, Finland
Hauptman-Woodward Medical Research Institute, Inc. (D.G.) Buffalo, New York 14203
Roswell Park Cancer Institute (D.G.) Buffalo, New York 14263

Human 17ß-hydroxysteroid dehydrogenase (17-HSD) type 1 catalyzes the conversion of the low activity estrogen, estrone, into highly active estradiol, both in the gonads and in target tissues. The present study was carried out to characterize the dimerization, microheterogeneity, and phosphorylation of human 17-HSD type 1 and to evaluate the current model of hydride transfer and substrate recognition of the enzyme, based on its x-ray structure. 17-HSD type 1 is a homodimer consisting of noncovalently bound subunits, and the data in the present study indicate an exceptionally strong association between the monomers [dissociation constant (K_d) < 5 pmol/liter]. Furthermore, substitutions constructed at the hydrophobic dimer interface always resulted in inactive aggregates of the protein. The enzyme was shown to be phosphorylated by protein kinase A exclusively at Ser¹³⁴ only in vitro. However, in contrast to previous suggestions, phosphorylation of Ser¹³⁴ was shown to play no role in the activity or microheterogeneity of human 17-HSD type 1. The presence of microheterogeneity in the recombinant enzyme also indicates that it does not result from the frequent protein polymorphism previously found for the enzyme. In line with the x-ray structure and the proposed catalytic mechanism of the enzyme, our results indicate that Ser¹⁴², Tyr¹⁵⁵, and Lys¹⁵⁹ are all critical for hydride transfer in human 17-HSD type 1. In contrast, the proposed interaction between His²²¹, Glu²⁸², and the 3-OH group of the steroid at the substrate recognition helix could not be shown to exist. Neither of these residues plays a critical role in the catalytic action of the enzyme in cultured cells.

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