title: An additional role for the Brønsted acid-base catalysts of mandelate racemase in transition state stabilization.
abstract: 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 pKa. 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 ΔCp 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University , Halifax, NS B3H 4R2, Canada.
authors.forename: Mitesh
authors.initials: M
authors.lastname: Nagar
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University , Halifax, NS B3H 4R2, Canada.|Department of Chemistry, Dalhousie University , Halifax, NS B3H 4R2, Canada.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Bearne
mesh_terms: D000143:Acids / Q000737:chemistry; D000595:Amino Acid Sequence; D002384:Catalysis; D019277:Entropy; D057927:Hydrophobic and Hydrophilic Interactions; D006877:Hydroxamic Acids / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016958:Pseudomonas putida / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000143:Acids; D006877:Hydroxamic Acids; D019998:Racemases and Epimerases; C021540:mandelate racemase