Mitochondrial 3?-hydroxysteroid dehydrogenase enzyme activity requires reversible pH-dependent conformational change at the intermembrane space.

ABSTRACT: The inner mitochondrial membrane protein 3?-hydroxysteroid dehydrogenase 2 (3?HSD2) synthesizes progesterone and androstenedione through its dehydrogenase and isomerase activities. This bifunctionality requires 3?HSD2 to undergo a conformational change. Given its proximity to the proton pump, we hypothesized that pH influences 3?HSD2 conformation and thus activity. Circular dichroism (CD) showed that between pH 7.4 and 4.5, 3?HSD2 retained its primarily ?-helical character with a decrease in ?-helical content at lower pH values, whereas the ?-sheet content remained unchanged throughout. Titrating the pH back to 7.4 restored the original conformation within 25 min. Metabolic conversion assays indicated peak 3?HSD2 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 3?HSD2 concentration from 1 to 40 ?g 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 3?HSD2 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, 3?HSD2 takes on a molten globule conformation that promotes the dual functionality of the enzyme.

SUBMITTER: Prasad M 

PROVIDER: S-EPMC3308737 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature