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

Abstract

Affinity labelling oft lie estradiol-l7β dehydrogenase of human placenta has been performed using derivatives of estradiol-17β 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 β 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