An Additional Role for the Brønsted Acid–Base Catalysts of Mandelate Racemase in Transition State Stabilization

• Published in: Biochemistry (Easton) Vol. 54; no. 44; pp. 6743 - 6752
• Main Authors: Nagar, Mitesh, Bearne, Stephen L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.11.2015
• Subjects: Acids - chemistry; Amino Acid Sequence; Catalysis; Entropy; Hydrophobic and Hydrophilic Interactions; Hydroxamic Acids - chemistry; Hydroxamic Acids - metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Pseudomonas putida - chemistry; Pseudomonas putida - enzymology; Pseudomonas putida - genetics; Pseudomonas putida - metabolism; Racemases and Epimerases - chemistry; Racemases and Epimerases - genetics; Racemases and Epimerases - metabolism; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.5b00982

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate and serves as a paradigm for understanding the enzyme-catalyzed abstraction of an α-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 Brønsted 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 benzohydroxamate (BzH) in an entropy-driven process with a value of ΔC 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 ∼2 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−π/NH−π 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 cyclohexanecarbohydroxamate revealed that cation−π/NH−π 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 Brønsted acid–base catalysts, and they stabilize both the enolate moiety and phenyl ring of the altered substrate in the TS.