Redefining the Minimal Substrate Tolerance of Mandelate Racemase. Racemization of Trifluorolactate Mitesh Nagar, † Ariun Narmandakh, † Yuriy Khalak, † and Stephen L. Bearne* ,†,‡ † Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada ‡ Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada ABSTRACT: Mandelate 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 β,γ-unsaturation is a requisite feature of substrates for the enzyme. We show that β,γ-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 α-postion of trifluorolactate using 1 H NMR spectrocsopy. β-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 ∼318-fold, resulting in catalytic efficiencies (k cat /K m ) that were ∼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 ΔH ‡ and ΔS ‡ 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 °C. Hence, mandelate racemase stabilizes the altered trifluorolactate in the transition state (ΔG tx ) by at least 20 kcal/mol. M andelate racemase (EC 5.1.2.2) is a member of the enolase superfamily of enzymes and catalyzes the Mg 2+ - dependent 1,1-proton transfer which interconverts the enantiomers of mandelate (Scheme 1). 1 The ability of MR to catalyze rapid carbon-hydrogen bond cleavage from a carbon acid with a relatively high pK a 2-4 makes it a useful paradigm for understanding enzyme-catalyzed proton abstraction from carbon acids. 1,4-7 Isotope exchange experiments, site-directed mutagenesis studies, and X-ray crystal structures of MR complexed with substrate and substrate analogues 8-12 indicate that catalysis proceeds via a two-base mechanism, with His 297 and Lys 166 abstracting the α-proton from (R)-mandelate and (S)-mandelate, respectively. 8,9,13 In addition, these experiments have revealed that Glu 317 acts as a general acid catalyst, 10 Lys 164 interacts with the carboxyl function of mandelate, 11 and Asn 197 interacts with the α-hydroxyl of mandelate to facilitate stabilization of the altered substrate in the transition state. 14 MR can utilize a variety of aryl- and heteroaryl-substituted mandelate derivatives as substrates. 15-20 To date, the simplest substrate identified is vinylglycolate. 21 The ability of MR to racemize this substrate and not ethylglycolate 21 and lactate 19 has suggested that β,γ-unsaturation is required for racemization. Indeed, crystal structures with bound atrolactate reveal that this substrate analogue assumes a conformation within the active site such that proton abstraction is stereoelectronically favored (i.e., the plane of the phenyl ring is approximately perpendicular to the C α -H bond, Figure 1). A comprehensive review of the substrate tolerance of MR conducted by Felfer et al. 15,22 led these authors to conclude that substrates of mandelate racemase must possess β,γ-unsaturation as a minimal requirement for activity. Herein, we show that the requirement for β,γ-unsaturation is not absolute and that stabilization of the negative charge of the enolic intermediate through inductive effects is also sufficient to promote racemization of a substrate. We show that MR accepts trifluorolactate (TFL) as a substrate and that the active site of the enzyme exhibits enhanced interaction with the trifluoromethyl group on the substrate relative to a methyl group. ■ METHODS AND MATERIALS (R)- and (S)-mandelic acid, (S)-trifluorolactic acid, and all other reagents, unless mentioned otherwise, were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). (R)-TFL was purchased from SynQuest (Alachua, FL). Circular dichroism (CD)-based assays and spectral measurements were conducted using a JASCO J-810 spectropolarimeter. NMR analyses were conducted at the Nuclear Magnetic Resonance Research Resource (NMR-3) using either a Bruker/Tecmag AC-250 spectrometer, Bruker AV-300 spectrometer, or Bruker AV-500 spectrometer. Enzyme Purification. Recombinant MR from Pseudomo- nas putida was overexpressed in and purified from Escherichia coli BL21(DE3) cells transformed with a pET-52b(+) plasmid Received: August 1, 2011 Revised: September 2, 2011 Published: September 6, 2011 Article pubs.acs.org/biochemistry © 2011 American Chemical Society 8846 dx.doi.org/10.1021/bi201188j | Biochemistry 2011, 50, 8846-8852