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-nitrophenoxide 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(-1) s(-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 pK(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 basecatalyzed 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.