MANDELATE RACEMASE: INSIGHTS INTO SUBSTRATE TOLERANCE, NOVEL INHIBITOR-BINDING MODES, AND THE ROLE OF BINDING DETERMINANTS

Abstract

Mandelate racemase (MR) is a useful model for studying the enzyme-catalyzed abstraction of an α-proton from carbon acid substrates with high pKa values. MR, an archetype of enolase superfamily, uses the Brønsted acid-base catalysts Lys 166 and His 297 in a two-base mechanism to catalyze the interconversion of the enantiomers of mandelic acid via an aci-carboxylate (enolate) intermediate. Based on the broad substrate spectrum of MR, it has been proposed that β,γ-unsaturation is a requisite feature of substrates. However, this thesis shows that MR can catalyze the interconversion of enantiomers of trifluorolactate with the chemical step being fully rate-limiting, suggesting that the presence of electron withdrawing groups such as the trifluoromethyl group on the α-carbon can stabilize the enolic intermediate through inductive effects. Thus, β,γ-unsaturation is not an absolute requirement for MR catalysis. A rather surprising finding was that the substrate-product analogue of trifluorolactate, 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)-propanoate (TFHTP), bound to MR with an affinity similar to that observed for transition state (TS) analogue inhibitors. Based on the x-ray crystal structure of the MR−TFHTP complex, the high binding affinity of TFHTP could be partly attributed to a novel binding-mode in which the carboxylate is involved in a salt-bridge with Lys 166 and His 297. Tartronate and mesoxalate were identified as reversible competitive inhibitors, which also form similar salt bridges, and 3-hydroxypyruvate acted as an irreversible, time-dependent inhibitor that undergoes Schiff-base formation with Lys 166 to form an aldehyde/enol(ate) adduct. Such an unprecedented reaction catalyzed by MR suggests a possible mechanistic link between the metal-dependent enolase superfamily and other α/β-barrel enzymes utilizing Schiff-base chemistry. The x-ray crystal structure of the MR-benzohydroxamate (BzH) complex suggested that Lys 166, His 297, and Tyr 54 play a role in BzH binding, and may afford TS stabilization. Tyr 54 mutants revealed that the role of Tyr 54 is relatively minor; however, isothermal titration calorimetry studies with the Lys 166- and/or His 297-MR mutants revealed that the Brønsted acid-base catalysts, especially Lys 166, interact with the hydroxamate moiety as well as the phenyl ring of BzH, possibly via cation-π/NH-π interactions. The work presented in this thesis suggests that the Brønsted acid-base catalysts can act as binding determinants and stabilize the enolate moiety of the altered substrate in the TS, a role that was previously overlooked.