Kuehm, Oliver2020-12-162020-12-162020-12-16http://hdl.handle.net/10222/80105Enzymes of the enolase superfamily (ENS) of share a high level of structural similarity and catalyze a conserved partial reaction yet display an overall mechanistic divergence. Mandelate racemase (MR) serves as a paradigm for the enzyme-catalyzed abstraction of an a-proton from a carbon acid substrate. This work described the characterization of a series of potent chloro- and fluoro-substituted phenylboronic acid (PBA) inhibitors of MR, including 3,4-dichloroPBA, for which MR exhibited the highest binding affinity observed to-date (Kd = 13.8 nM). Investigations on the conserved KxK motif of the MR subgroup, revealed that the KxK residue was essential for catalysis and for the stability of MR and L-tartrate dehydratase. The K164M and K164R MR variants displayed a change in thermal melting temperature (∆Tm) of –4.03 and –8.36 °C, respectively. Finally, approaches towards production of human reverse thymidylate synthase, an ENS enzyme, using precursor proteins bearing two protease specific sites were described.enEnolaseMandelate racemaseInhibitionThermal stabilityReverse thymidylate synthaseThesis