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[摘要]：Mande late racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the enantiomers of mandelic acid and a variety of aryl- and heteroaryl-substituted mandelate derivatives, suggesting that beta,gamma-unsaturation is a requisite feature of substrates for the enzyme. We show that beta,gamma-unsaturation is not an absolute requirement for catalysis and that mandelate racemase can bind and catalyze the racemization of (S)-trifluorolactate (k(cat) = 2.5 +/- 0.3 s(-1), K(m) = 1.74 +/- 0.08 mM) and (R)-trifluorolactate (k(cat) = 2.0 +/- 0.2 s(-1), K(m) = 1.2 +/- 0.2 mM). The enzyme was shown to catalyze hydrogen-deuterium exchange at the alpha-postion of trifluorolactate using (1)H NMR spectrocsopy. beta-Elimination of fluoride was not detected using (19)F NMR spectroscopy. Although mandelate racemase bound trifluorolactate with an affinity similar to that exhibited for mandelate, the turnover numbers (k(cat)) were markedly reduced by similar to 318-fold, resulting in catalytic efficiencies (k(cat)/K(m)) that were, similar to 400-fold lower than those observed for mandelate. These observations suggested that chemical steps on the enzyme were likely rate-determining, which was confirmed by demonstrating that the rates of mandelate racemase-catalyzed racemization of (S)-trifluorolactate were not dependent upon the solvent microviscosity. Circular dichroism spectroscopy was used to measure the rates of nonenzymatic racemization of (S)-trifluorolactate at elevated temperatures. The values of Delta(double dagger)Ht and Delta S(double dagger) for the nonenzymatic racemization reaction were determined to be 28.0 (+/- 0.7) kcal/mol and -15.7 (+/- 1.7) cal K(-1) mol(-1), respectively, corresponding to a free energy of activation equal to 33 (+/- 4) kcal/mol at 25 degrees C. Hence, mandelate racemase stabilizes the altered trifluorolactate in the transition state (Delta G(tx)) by at least 20 kcal/mol. |
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