Mandelate Racemase in Pieces: Effective Concentrations of Enzyme Functional Groups in the Transition State

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Abstract
Mandelate racemase (EC 5.1.2.2) catalyzes the abstraction of a proton from a carbon atom adjacent to a carboxylate function, a reaction which is kinetically and thermodynamically unfavorable. Proton NMR spectroscopy and polarimetry were used to measure the rates of deuterium incorporation into the α-position of mandelate and the racemization of (R)-mandelate, after samples had been incubated at elevated temperatures. Using an Arrhenius plot, the value of the free energy of activation for racemization and deuterium exchange was calculated to be 34.6 (±0.9) kcal/mol under neutral conditions, at 25 °C. This result indicates that mandelate racemase produces a remarkable rate enhancement [(1.7 × 1015)-fold], and a level of transition state affinity (Ktx = 2 × 10-19 M), that surpasses the levels achieved by most enzymes. Methylamine, imidazole, and acetate catalyzed the nonenzymatic hydrogen−deuterium exchange reaction at 170 °C, and the values of the second-order rate constants are 2.8 (±0.2) × 10-5, 13.4 (±0.7) × 10-5, and ≤4 (±1) × 10-7 M-1 s-1, respectively. By comparing wild-type mandelate racemase's proficiency as a catalyst with the proficiencies of these small molecules which correspond to the missing pieces in the variant enzymes Lys166Arg [Kallarakal, A. T., et al. (1995) Biochemistry34, 2788−2797], His297Asn [Landro, J. A., et al. (1991) Biochemistry30, 9274−9281], and Glu317Gln [Mitra, B., et al. (1995) Biochemistry34, 2777−2787], we estimate the effective concentrations of the catalytic side chains of Lys 166, His 297, and Glu 317 as ≥622, ≥3 × 103, and ≥3 × 105 M, respectively, in the native protein. These observations support the view that general acid−general base catalysis, inefficient in simple model systems, becomes an efficient mode of catalysis when structural complementarity between an enzyme and its substrate is optimized in the transition state.