Affinity Labelling of the Estradiol‐17β Dehydrogenase from Human Placenta with Substrate Analogs

Abstract

Affinity labelling of the estradiol-178 dehydrogenase of human placenta has been performed using derivatives of estradiol-17P carrying alkylating groups in nine different positions on the steroid nucleus.The active-site-directed character of the inhibitions is confirmed by the following observations : the affinity labels are substrates or competitive inhibitors, the enzyme is protected against inactivation and alkylation by the substrate and by the coenzyme, the stoichiometry of the alkylation is two moles of inhibitor per 68000 g of enzyme (dimer).The alkylation of a histidine residue which is fast and extensive when the alkylating side chain is on the C-3 carbon atom, is dramatically decreased when the alkylating side chain is shifted towards rings B and D. These results allow the location of this histidine in the vicinity of ring A and probably on the P face of the steroid nucleus.The reactivity of a cysteine located on the active site was quite different, showing increasing alkylation when the alkylating substituent of the affinity labels was shifted from C-3 to C-16 of the steroid nucleus.The correlation of this result and that obtained using an alkylating analog of NAD (3-chloroacetylpyridine-adenine dinucleotide) indicates that this cysteine is located in the catalytic region of the active site, at the junction of the ring D of the steroid nucleus with the nicotinamide moiety of the coenzyme.Affinity labelling of steroid dehydrogenases was performed by Warren [I-31 on the bacterial 20p-hydroxysteroid dehydrogenase, by Engel [4,5] and by ourselves [6-81 on the estradiol-17P dehydrogenase of human placenta.This estradiol dehydrogenase (B enzyme) transfers the 4 -p r o 3 hydrogen atom of nicotinamide to 17-0x0-steroids which are converted into 17~-hydroxysteroids (S alcohols) [9,10]. This stereospecificity is strict and implicates, according to Prelog [ll], that during the transition state, the CI face of the substrate is always presented to the B face of nicotinamide. This mechanistic feature and the relative rigidity of the substrate molecule make estradiol-178 dehydrogenase an enzyme very suitable for affinity labelling : the substrate binding site can be studied using several substrate analogs, each carrying an alkylating substituent in a ~ Enzyme. EstradiolLl7fi dehydrogenase (EC 1.1.1.62). Trivial names. 16a-Broinoacetoxy-estrone 3-acetate, 17-0x0-1,3,5( lO)-estratrien-3,16cc-dio~ 3-acetate 16-bromoacetate; I6c-bromoacetoxy-estradiol3-methyl ether, 3-methoxy-I ,3,5( 10)-estratrien16a,l7p-diol 16-broinoacetate; 3-iodoacetoxy-estrone, 17-oxo-1,3,5-(1 O)-estratrien-3-yl iodoacetate ; 3-iodoacetoxy-estradiol, 17/M~y-droxy-l,3,5(10)-estratrien-3-yI iodoacetate. different position : under such conditions the region to be explored can be selected by the position and the orientation of the alkylating group on the steroid nucleus (Fig. 1). Thus, the localization of labelled amino acid in the active site can be performed with some accuracy, provided the orientation of the substrate analog during alkylation is i...

Affinity Labeling of Arginyl Residues at the Catalytic Region of Estradiol 17β‐Dehydrogenase from Human Placenta by 16‐Oxoestrone

“…Crastes de Paulet et al [26] reported that the histidyl residue was located also in vicinity of the A-ring, while one cysteinyl residue was located in the catalytic region, the second one was also located in the cofactor-binding site, and third one was probably present outside of the active-site. Also, Chin et al reported that two different histidyl residues were present in the dehydrogenase 1271, one histidyl residue was located in the catalytic region close to the D-ring of the bound steroid [24] and the other was in the vicinity of its A-ring [22]. In addition to those findings, estradiol 17P-dehydrogenase of human placenta has 22 residues of arginine per subunit (Mr = 34000) [23].…”

“…On the other hand, the three nucleophilic amino acids, such as histidyl, cysteinyl, and lysyl residues, were identified in the active-site of estradiol 17j-dehydrogenase by the affinity labeling with haloacetoxylated ligands of the substrate [22 -241 and cofactor [22] derivatives. Lysyl residue was located in the vicinity of A-ring of the bound steroid, but was not essential for the enzyme activity itself [25].…”

La 17ß Hydroxystéroïde Déshydrogénase Sérique et Tissulaire et les Œstrogènes dans la Môle Hydatiforme

“…Depuis sä decouverte dans le tissu placentaire humain (11), la 17ß hydroxystoro'ide doshydrogenase figure parmi les enzymes les mieüx etudiees du metabolisme hormonal steroi'dien (12)(13)(14)(15) (16) a note une activite anormalement forte de la 77ß hydroxystero'ide deshydrogonase serique, nous confirmons ce phenomene. Toutefois, aux stades precoces de Faffection que l'auteur n'avait pu explorer, nous constätons que Factivite enzymatique reste dans la fourchette des valeurs enfegistrees pour des grossesses normales de meme äge (9 ä 10 semaines).…”

“…Prices are subject to change without notice Prices are subject to change without notice (14) En tube de gel de polyacrylamide, le m61ange colorant revfcle apres migration, une seule bände avec les extraits molaires, cette bände est retrouvee avec le tissu placentaire. Cependant, ä partir de ce dernier tissu, trois autres bandes color6es sont observees, dues vraisemblablemeiit ä d'autres deshydrogonases, differentes de la 17ß hydroxystoro'ide deshydrogenase, car elles apparaissent en l'absence d'cestradiol.…”

Effects of ethanol and other organic solvents on the kinetic behaviour of purified human placental oestradiol-17β-dehydrogenase

“…We have presented an example of the profound effects that organic solvents can have on kinetic parameters. As already mentioned in the introduction, Chin & Warren (1975) and Pons et al (1976) obtained different results in their affinity-labelling experi¬ ments of human placental oestradiol dehydrogen-ase when using different co-solvents in water. 1980).…”