An additional role for the Brønsted acid-base catalysts of mandelate racemase in transition state stabilization.
Biochemistry
; 54(44): 6743-52, 2015 Nov 10.
Article
en En
| MEDLINE
| ID: mdl-26480244
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 â S reaction direction. In the S â 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 â¼2 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.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Pseudomonas putida
/
Racemasas y Epimerasas
/
Ácidos Hidroxámicos
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Biochemistry
Año:
2015
Tipo del documento:
Article
País de afiliación:
Canadá