Structure-reactivity relationships for β-galactosidase (escherichia coli, lac Z): A second derivative effect on β_nuc for addition of alkyl alcohols to an oxocarbenium ion reaction intermediate

Velocities for the synthesis of trifluoroethyl 2-deoxy-β-D- galactopyranoside by transfer of the 2-deoxygalactosyl group from β-galactosidase to trifluoroethanol were determined from studies of the β-galactosidase-catalyzed cleavage of 4-nitrophenyl-2-deoxy-β-D- galactopyranoside as the difference in rates of appearance of 4-nitrophen-oxide anion and 2-D-deoxygalactose. These data were used to calculate a rate constant ratio of k_ROH/k_s = 2.3 M^-1 for partitioning of the intermediate between addition of trifluoroethanol and solvent water. Velocities for the synthesis of other alkyl 2-deoxy-β-D-galactopyranosides by transfer of the 2-deoxygalactosyl group from β-galactosidase to alkyl alcohols were determined from the effect of alkyl alcohols on the velocity of β-galactosidase-catalyzed cleavage of 4-nitrophenyl-2-deoxy-β-D- galactopyranoside in a reaction where breakdown of the intermediate is rate determining. These data were used to calculate rate constant ratios k _ROH/k_s for the reactions of eight alkyl alcohols. Absolute rate constants k_ROH (M^-1S^-1) were calculated from k_ROH/k_s and k_s = 0.002 s^-1 for the addition of water. A Brønsted coefficient of β_nuc = -0.07 ± 0.08 was determined as the slope of a logarithmic correlation of k_ROH against alcohol p_a. The change from a 2-OH to a 2-H substituent at the β-D-galactopyranosyl intermediate causes a 0.12 ± 0.04 increase in the value of β_nuc for alcohol addition. This anti-Hammond effect provides evidence that general base-catalyzed addition of alcohols to an enzyme-bound β-D-galactopyranosyl oxocarbenium ion intermediate proceeds along a reaction coordinate in which there is strong coupling between carbon-oxygen bond formation and proton transfer from the alcohol to a basic residue at the enzyme.