Estradiol 17 -dehydrogenase

In a very different enzyme system, Auchus and Covry used 13C NMR to demonstrate that the NAD+-dependent estradiol dehydrogenase from human placenta was inactivated by acetylenic steroids via Michael addition of an enzymic LysNH2 to the acetylenic ketosteroid generated during the reaction164 and thereby forming an en-aminone (equation 26). 

The endogenous oestrogens, for instance estrone and 17P-estradiol are observed to be interconvertible biochemically in the presence of the specific enzyme estradiol dehydrogenase and... 

Importantly, these hormones including estradiol dehydrogenase are chiefly metabolized in the liver and mostly get excreted as water-soluble glucuronide and sulphate conjugates. Estrone is regarded as a less active (1/12) metabolite of estradiol. 

Fig. 45.3. Biosynthesis of androgens and other sex steroid hormones. The enzymes involved in this biosynthesis are (a) side-chain cleavage, (b) 17a-hydroxylase (c) 5-ene-3(3-hydroxysteroid dehydrogenase, (d) 3-oxosteroid-4,5-isomerase, (e) 17,20-lyase, (f) 17(3-hydroxysteroid dehydrogenase, (g) aromatase, (h) estradiol dehydrogenase, and (i) 5a-reductase.

Isolation and cloning of rat MFP-2" revealed its identity to an enzyme called type IV np-estradiol dehydrogenase. Independently, Hiltunen and coworkers, who also confirmed the D-specificity of the protein, and Gustafsson and coworkers also obtained the rat cDNA. Type IV 17P-estradiol dehy drogenase was isolated initially from porcine endometrium and subsecprently cloned by Adamski and coworkers. " The recombinant porcine enzyme displays crotonase and 3-hydroxybutyryl-CoA dehydrogenase activities ... 

Leenders, R, Husen, B., Thole, H.H. Adamski, J. (1994) ATo/. Cell. Endocrinol. 104, 127-131. The sequence of porcine 80kDa 17 p-estradiol dehydrogenase reveals similarities to the short chain alcohol dehydrogenase family, to actin binding motifs and to sterol carrier protein 2. 

Markus, M., Husen, B., Leenders, F., Jungblut, P.W., Hall, P.F. Adamski, J. 9)S)Eur. J. Cell. Biol 68, 26i-261. The organelles containing porcine 17 beta-estradiol dehydrogenase are peroxisomes. 

We have studied the reaction of the potential afiSnity label, 3-bromo-acetoxyestrone, with human placental estradiol dehydrogenase and have found that it satisfies the first four criteria for an afiSnity label. Furthermore, the 17-keto group of this compound offers an opportunity to determine whether, after reaction with the enzyme, the covalently bound steroid is suitably positioned to serve as a substrate in the dehydrogenase reaction. We provide evidence that such is indeed the case. 

We have reported that bromoacetoxyestrone is a substrate for estradiol dehydrogenase. Bromoacetoxyestrone inactivates this enzyme with... 

In these experiments, 100% inactivation was not achieved native enzyme was, therefore, always present. Consequently, it was necessary to determine whether the reduction of estrone was catalyzed by residual native enzyme acting upon enzyme-bound steroid [Eq. (1)] or by modified enzyme acting upon its own covalently bound steroid [Eq. (2)]. These alternatives may be distinguished by exploiting the transhydro-genase function of this enzyme and the fact that diethylstilbestrol inhibits estradiol dehydrogenase and its transhydrogenase function. 

In the transhydrogenase system, all components for the assay are present except estradiol dehydrogenase and steroid. Addition of aflSnity labeled estradiol dehydrogenase to the assay mixture simultaneously introduces the potential catalytic agents, enzyme (either native or modified) and steroid (estrone covalently linked to estradiol dehydrogenase). 

These data show that the steroid molecule in the labeled estradiol dehydrogenase is capable of assuming the geometric relations to enzyme and cofactor that are required for the catalytic event to occur. The affinity label is therefore catalytically competent. 

---