An Additional Role for the Bronsted Acid-Base Catalysts of Mandelate Racemase in Transition State Stabilization

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate and serves as a paradigm for understanding the enzyme-catalyzed abstraction of an a-proton from a carbon acid substrate with a high pK(a). The enzyme utilizes a two-base mechanism with Lys 166 and His 297 acting as Bronsted acid and base catalysts, respectively, in the R -> S reaction direction. In the S ->) R reaction direction, their roles are reversed. Using isothermal titration calorimetry (ITC), MR is shown to bind the intermediate/transition state (TS) analogue inhibitor benzo-hydroxamate (BzH) in an entropy-driven process with a value of Delta C-p, equal to -358 +/- 3 cal mol(-1) K-1, consistent with an increased number of hydrophobic interactions. However, MR binds BzH with an affinity that is orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions [St. Maurice, M., and Bearne, S. L. (2004) Biochemistry 43, 2524], suggesting that additional specific interactions contribute to binding. To test the hypothesis that cation-pi/NH-pi interactions between the side chains of Lys 166 and His 297 and the aromatic ring and/or the hydroxamate/hydroximate moiety of BzH contribute to the binding of BzH, site-directed mutagenesis was used to generate the MR variants K166M, K166C, H297N, and K166M/H297N and their binding affinity for various ligands determined using ITC. Comparison of the binding affinities of these MR variants with the intermediate/TS analogues BzH and cydohexanecarbohydroxamate revealed that cation-pi/NH-pi interactions between His 297 and the hydroxamate/hydroximate moiety and the phenyl ring of BzH contribute approximately 0.26 and 0.91 kcal/mol to binding, respectively, while interactions with Lys 166 contribute approximately 1.74 and 1.74 kcal/mol, respectively. Similarly, comparison of the binding affinities of these mutants with substrate analogues revealed that Lys 166 contributes >2.93 kcal/mol to the binding of (R)-atrolactate, and His 297 contributes 2.46 kcal/mol to the binding of (S)-atrolactate. These results are consistent with Lys 166 and His 297 playing dual roles in catalysis: they act as Bronsted acid-base catalysts, and they stabilize both the enolate moiety and phenyl ring of the altered substrate in the TS.