Estradiol 17β-dehydrogenase: full enzymatic activity in the absence of zinc

Abstract

The precise catalytic mechanism of the steroid interconverting enzyme, human placental estradiol 17β-dehydrogenase (EC 1.1.1.62, estradiol-17β:NAD +17-oxidoreductase), is not known. Two general models for the catalytic mechanism of dehydrogenase have been defined. One model requires Zn 2+ metal for the catalytic event, as has been shown for horse liver alcohol dehydrogenase (EC 1.1.1.1, alcohol: NAD + oxidoreductase). Another model has been demonstrated for the 2-hydroxy acid dehydrogenases in which histidine residues are necessary for enzyme activity, without participation of a metal ion. In order to define which mechanism might be operative for the placental enzyme, it became important to determine whether Zn 2+, or another metal ion, is associated with the macromolecule. Several homogeneous enzyme preparations, having protein concentrations from 5–80 μM, were extensively dialyzed in a buffer containing EDTA. Atomic absorption analysis of each sample demonstrated that no Zn 2+ was present, although the enzymatic activity was maintained. In addition, there was no significant detection of Mg 2+ or Mn 2+ above background levels. When the isolated enzyme was dialyzed against buffer containing added 0.01–20 μM ZnCl 2, no increase in specific activity of the enzyme was seen. The data indicate that the presence of zinc is not required for the catalytic event. These results, together with our previous affinity-labeling studies, which demonstrate a histidine residue in the catalytic region of the active site, allow us to propose that the catalytic mechanism of the human placental estradiol 17β-dehydrogenase is similar to that of the 2-hydroxy acid dehydrogenases.