Human placental estradiol 17β-dehydrogenase: structural and catalytic changes during urea denaturation

Abstract

The denaturation behavior of human placental estradiol 17β-dehydrogenase (EC 1.1.1.62) in urea was studied by following changes in enzyme activity, conformation and oligomeric state. Results showed that the native → unfolded transition follows a complex pattern, in which changes in both secondary and tertiary structure are simultaneous with changes in the aggregation state of enzyme. At relatively low urea (< 3 M), a major conformational transition, as monitored by CD and fluorescence measurements, is concomitant with an expanded state of the enzyme that coincides with its inactivation and the formation of polymeric species. Protein structural changes were also monitored by using the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid. The combined data suggest the existence of a molten globule state of dimeric enzyme promoted by low urea concentrations. Dilution of urea at this stage results in a full recovery of the enzymatic activity as well as of the native dimeric structure. Between 3 and 5 M urea estradiol 17β-dehydrogenase exists as a mixture of high molecular mass species which may be resolved by electrophoresis. In this range of urea concentration, only minor conformational changes were detected, although inactivation becomes to be irreversible. Above 5 M urea a second conformational transition takes place. Electrophoretic analysis of cross-linked samples revealed this stage results in the complete dissociation of enzyme toward unfolded monomer. It is concluded that the inactivation and unfolding of estradiol 17β-dehydrogenase during denaturation by urea occurs with the formation of intermediate species with different stability in which a molten globule-like state appears to be involved. The irreversibility of the process above urea 3 M is explained as the inability of aggregated enzyme to dissociate into native dimers.