title: Mitochondrial 3β-hydroxysteroid dehydrogenase enzyme activity requires reversible pH-dependent conformational change at the intermembrane space.
abstract: The inner mitochondrial membrane protein 3β-hydroxysteroid dehydrogenase 2 (3βHSD2) synthesizes progesterone and androstenedione through its dehydrogenase and isomerase activities. This bifunctionality requires 3βHSD2 to undergo a conformational change. Given its proximity to the proton pump, we hypothesized that pH influences 3βHSD2 conformation and thus activity. Circular dichroism (CD) showed that between pH 7.4 and 4.5, 3βHSD2 retained its primarily α-helical character with a decrease in α-helical content at lower pH values, whereas the β-sheet content remained unchanged throughout. Titrating the pH back to 7.4 restored the original conformation within 25 min. Metabolic conversion assays indicated peak 3βHSD2 activity at pH 4.5 with ~2-fold more progesterone synthesized at pH 4.5 than at pH 3.5 and 7.4. Increasing the 3βHSD2 concentration from 1 to 40 μg resulted in a 7-fold increase in progesterone at pH 4.5, but no change at pH 7.4. Incubation with guanidinum hydrochloride (GdmHCl) showed a three-step cooperative unfolding of 3βHSD2 from pH 7.4 to 4.5, possibly due to the native state unfolding to the intermediate ion core state. With further decreases in pH, increasing concentrations of GdmHCl led to rapid two-step unfolding that may represent complete loss of structure. Between pH 4 and 5, the two intermediate states appeared stable. Stopped-flow kinetics showed slower unfolding at around pH 4, where the protein is in a pseudostable state. Based on our data, we conclude that at pH 4-5, 3βHSD2 takes on a molten globule conformation that promotes the dual functionality of the enzyme.
authors.affiliation: Mercer University School of Medicine, Savannah, Georgia 31404, USA.
authors.forename: Manoj
authors.initials: M
authors.lastname: Prasad
authors.forename: James L
authors.initials: JL
authors.lastname: Thomas
authors.forename: Randy M
authors.initials: RM
authors.lastname: Whittal
authors.forename: Himangshu S
authors.initials: HS
authors.lastname: Bose
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000201:enzymology*; D011375:Progesterone Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: C533068:3 beta-hydroxysteroid dehydrogenase type II; D011375:Progesterone Reductase