HUMAN PLACENTAL ESTRADIOL 17-BETA-DEHYDROGENASE - STRUCTURAL AND CATALYTIC CHANGES DURING UREA DENATURATION

The denaturation behavior of human placental estradiol 17 beta-dehydro genase (EC 1.1.1.62) in urea was studied by following changes in enzym e activity, conformation and oligomeric state. Results showed that the native --> unfolded transition follows a complex pattern, in which ch anges in both secondary and tertiary structure are simultaneous with c hanges in the aggregation state of enzyme. At relatively low urea (<3 M), a major conformational transition, as monitored by CD and fluoresc ence measurements, is concomitant with an expanded state of the enzyme that coincides with its inactivation and the formation of polymeric s pecies. Protein structural changes were also monitored by using the hy drophobic probe 1-anilinonaphthalene-8-sulfonic acid. The combined dat a suggest the existence of a molten globule state of dimeric enzyme pr omoted by low urea concentrations. Dilution of urea at this stage resu lts in a full recovery of the enzymatic activity as well as of the nat ive dimeric structure, Between 3 and 5 M urea estradiol 17 beta-dehydr ogenase exists as a mixture of high molecular mass species which may b e resolved by electrophoresis. In this range of urea concentration, on ly minor conformational changes were detected, although inactivation b ecomes to be irreversible. Above 5 M urea a second conformational tran sition takes place. Electrophoretic analysis of cross-linked samples r evealed this stage results in the complete dissociation of enzyme towa rd unfolded monomer. It is concluded that the inactivation and unfoldi ng of estradiol 17 beta-dehydrogenase during denaturation by urea occu rs with the formation of intermediate species with different stability in which a molten globule-like state appears to be involved. The irre versibility of the process above urea 3 M is explained as the inabilit y of aggregated enzyme to dissociate into native dimers.