The Impact of Bilirubin on 7α- and 7β-Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

Abstract

7α- and 7β-hydroxysteroid dehydrogenases (HSDHs) are enzymes that can catalyze the isomerization of hydroxyl groups at site seven of bile acids. In a previous study, we found that the activities of 7α- and 7β-HSDHs can be inhibited by bilirubin. In order to clarify the impact, the effects of bilirubin on enzymes were studied by kinetics, spectrum, and docking analysis. The relative activity of 7α-HSDH remained less than 40% under 1 mM bilirubin, and only 18% activity of 7β-HSDH kept in the same condition. Using taurochenodeoxycholic acid (TCDCA) as substrate, the Km of 7α-HSDH was up to 0.63 mM from 0.24 mM after binding with bilirubin and the Km of 7β-HSDH rose from 1.14 mM to 1.87 mM for the catalysis of tauroursodeoxycholic acid (TUDCA). The affinity of 7α- and 7β-HSDHs to substrates decreased with the effect of bilirubin. The binding of bilirubin with 7α- or 7β-HSDHs was analyzed by UV–vis, fluorescence, and circular dichroism (CD) spectroscopy. The results reflected that bilirubin caused a slight change in the secondary structure of 7α- or 7β-HSDHs, and the changes were correlated with the ratio of bilirubin to enzymes. Ten candidate molecular docking results were presented to reflect the binding of bilirubin with 7α- or 7β-HSDHs and to explore the inhibition mechanism. This research provides a more in-depth understanding of the effect of bilirubin on 7α- and 7β-HSDHs.