Enzyme, Cosolvent, and Substrate Interactions in PLE Hydrolysis Reactions and Evaluation of the Stability of an Unnatural Glutathione Analogue

Prochiral diester malonates have been hydrolyzed in the presence of Pig Liver Esterase (PLE). Several of the diesters produced the respective half-ester in moderate to high enantioselectivity. A series of cosolvent assays were performed to evaluate the ability of the cosolvent to influence the enantioselective outcome of the hydrolysis reactions. Ethanol produced the largest changes in enantioselectivity of all solvents evaluated. The isoenzymes of PLE were also evaluated and provided very different enantioselective outcomes than that of crude PLE. A series of NMR titrations was performed to explore the interactions between the substrates and ethanol cosolvents. Improvements to our previously reported mass spectrometry assay for the determination of the enantioselectivity of the PLE hydrolysis reaction are discussed. The improvements to this assay will allow for the determination of both yield and enantioselectivity of the PLE hydrolysis reaction in a single analysis. A glutathione analogue containing α-methyl cysteine has been evaluated for inhibitory activity against the enzyme glutathione reductase (GR). The methylated glutathione analogue (mGSSG) has been found to be an inhibitor of the GR enzyme. Molecular modeling studies suggest that the disulfide bond in the mGSSG is twisted in comparison to the natural GSSG