Effects of bile salts on the solubility and activity of yeast alcohol dehydrogenase in AOT reversed micelles

The effects of two bile salts, sodium taurocholate (NaTC) and 3-[(3-cholamidylpropyl) dimethylammonio]-1-propane sulfonate (CHAPS), on the solubility, stability and catalytic activity of yeast alcohol dehydrogenase (YADH) in bis(2-ethylhexyl) sodium sulfosuccinate (AOT) reversed micelles are reported. Spectroscopic measurements of extrinsic fluorescent probes and intrinsic fluorescent residues of YADH indicate that adding bile salt to AOT reversed micelles changes the relative polarity of the interfacial region and the rotational freedom of the solubilized enzyme. Activity measurements were performed using ethanol as the substrate and β-nicotinamide adenine dinucleotide (β-NAD) as the coenzyme. YADH activity in the reversed micelles is optimal at pH 8.0 and increases with increasing ω (ω=[water]/[AOT]), presumably due to larger water pools in the micellar interior. Activity is lower in the AOT micelles than in buffer solution but some activity is regained upon addition of bile salt. Addition of bile salt also increases the stability of YADH in the reversed micelles. Kinetic analysis of the enzymatic reaction data shows that YADH has a lower affinity for ethanol in reversed micelles than in buffer, likely due to partitioning of the ethanol among the different phases in the reversed micellar media. Bile salts increase both the affinity of YADH for ethanol and the reaction velocity in the reversed micelles. The effects of bile salt are attributed primarily to an increase in the size of the water pool in the reversed micelle in the presence of bile salt, which allows the enzyme to assume a more active conformation.