Mandelate racemase in pieces: Effective concentrations of enzyme functional groups in the transition state

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 ct-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 degrees C. This result indicates that mandelate racemase produces a remarkable rate enhancement [(1.7 x 10(15))fold], and a level of transition state affinity (K-tx = 2 x 10(-19) M), that surpasses the levels achieved by most enzymes. Methylamine, imidazole, and acetate catalyzed the nonenzymatic hydrogen-deuterium exchange reaction at 170 degrees C, and the values of the second-order rate constants are 2.8 (+/-0.2) x 10(-5), 13.4 (+/-0.7) x 10(-5), and less than or equal to 4 (+/-1) x 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) Biochemistry 34, 2788-2797], His297Asn [Landro, J. A., et al. (1991) Biochemistry 30, 9274-9281], and Glu317Gln [Mitra, B., et al. (1995) Biochemistry 34, 2777-2787], we estimate the effective concentrations of the catalytic side chains of Lys 166, His 297, and Glu 317 as greater than or equal to 622, greater than or equal to 3 x 10(3), and greater than or equal to 3 x 10(5) 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.