Allosteric interaction between 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase and cytochrome b5 influences cofactor binding.

The biosynthesis of steroid hormones, essential to the stwvival of all mammals, is dependent on the activity of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3βBHSD). 3βHSD activity is, in turn, influenced by cytochrome-b5 (Cyt-b5). However, the mechanism through which this occurs is unknown. In this study, we investigated this mechanism by evaluating the influence of Cyt-b5 on the dehydrogenase and isomerase activities of 3βHSD. Capra hircus 3IBHSD was overexpressed in SF-9 cells, using a baculovirus expression system, and purified. Substrate and cofactor kinetics were determined spectrophotometrically in the presence and absence of purified Ovis aries liver Cyt-b5. Nonspecific enzyme activity was evaluated by zero-enzyme, -substrate, and -cofactor blanks. Fusion proteins, 3βHSD-eCFP, and Cyt-b5-eYFP were subsequently coexpressed in COS-1 cells and analyzed for FRET. A CFP-YFP fusion protein served as positive control, while coexpression of 3βHSD- eCFP and cytochrome P450 17α-hydroxylase/17,20 lyase-eYFP (CYP17AI-eYFP) served as negative control. Results showed Cyt-b5 to decrease the Km,NAD+ value of 3βHSD ≈3.5-fold while increasing the Vmax,app of the dehydrogenase reaction ≈17%. FRET analysis showed COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b5-eYFP to exhibit a FRET signal ≈9-fold greater than that of the negative control. These results indicate that Cyt-b5 augments 3βHSD activity v/a an allosteric mechanism by increasing the affinity of the enzyme toward NAD+. [ABSTRACT FROM AUTHOR]