12α-hydroxysteroid dehydrogenase (12α-HSDH) from Methanosphaera Stadtmanae catalyzes the breakdown of deoxycholic acid into 12-oxo-lithocholic acid, which is important as the buildup of deoxycholic acid can lead to issues such as colon cancer. In this project, inhibition and ligand binding profiles of 12α-HSDH with NAD are being explored. Previously, we did substrate specificity experiments and found that 12α-HSDH is strictly NADP⁺ dependent, therefore, interference of NADP⁺ to the enzyme binding would cripple the reaction. NAD⁺, being a structural analogue of NADP⁺, was chosen to attempt the inhibition studies.
12α-HSDH is overexpressed from a recombinant plasmid in E. coli and purified using immobilized metal affinity chromatography. Preliminary experimental data revealed that NAD⁺ exhibits inhibitory properties for 12α-HSDH’s reaction. The end-point assays are monitored spectrophotometrically at 340 for the conversion of NADP⁺ to NADPH without and with variable NAD⁺ concentrations.
Next, steady-state kinetics will be performed for variable DCA concentrations with increasing concentrations of NAD⁺ to obtain the multiple Michaelis Menten and Lineweaver Burk plots. The inhibition constant of NAD⁺ as well as type of inhibition will be determined for the reaction. The inhibition type can be deduced from the spot of intersection of lines on the Lineweaver Burk plot. Also, for different types of inhibition patterns, kinetic parameters Michaelis Menten constant and maximal velocity may or may not change in the presence of an inhibitor, telling us more about the inhibition type. To supplement the inhibition studies, ligand binding studies will be conducted using tyrosine fluorescence quenching to obtain the dissociation constant values for NADP⁺ and NAD⁺. Also, it will help us to compare the affinities of substrate and inhibitor for the enzyme.

Marshall Ritchie

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