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 whic h is kinetically and thermodynamically unfavorable. Proton NMR spectro scopy and polarimetry were used to measure the rates of deuterium inco rporation 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 i ndicates that mandelate racemase produces a remarkable rate enhancemen t [(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 hydrog en-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), res pectively. By comparing wild-type mandelate racemase's proficiency as a catalyst with the proficiencies of these small molecules which corre spond to the missing pieces in the variant enzymes Lys166Arg [Kallarak al, A. T., et al. (1995) Biochemistry 34, 2788-2797], His297Asn [Landr o, J. A., et al. (1991) Biochemistry 30, 9274-9281], and Glu317Gln [Mi tra, B., et al. (1995) Biochemistry 34, 2777-2787], we estimate the ef fective concentrations of the catalytic side chains of Lys 166, His 29 7, 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 genera l acid-general base catalysis, inefficient in simple model systems, be comes an efficient mode of catalysis when structural complementarity b etween an enzyme and its substrate is optimized in the transition stat e.