Effect of an E461G Mutation of β-Galactosidase (Escherichia coli, lac Z) on pL Rate Profiles and Solvent Deuterium Isotope Effects

An E461G mutation of β-galactosidase results in the disappearance of the high pL (L = H, D) downward break in the rate profiles for kcat/Km for wild-type enzyme-catalyzed hydrolysis of 4-nitrophenyl β-d-galactopyranoside (Gal-OPNP) and a decrease from (kcat)HOH/(kcat)DOD = 1.7 to (kcat)HOH/(kcat)DOD = 1.2 in the solvent deuterium isotope effect. These observations provide evidence that the propionic acid side chain of Glu 461 is protonated at catalytically active free β-galactosidase and they are consistent with a role for this residue in Brønsted acid catalysis at the leaving group. The earlier observation that this same E461G mutation results in the loss of a downward break at high pH in the rate profile for ks for transfer of the β-d-galactopyranosyl group from β-galactosidase to water cannot be simply explained by a mechanism in which the single side chain of Glu 461 functions to provide general acid catalysis in the rate limiting step for formation of the β-d-galactopyranosyl intermediate and general base catalysis of breakdown of this intermediate. Evidence is presented that there may be different catalytic mechanisms, with different roles for the side chain for Glu-461, for nucleophilic addition of water and of small alkyl alcohols to the β-d-galactopyranosyl reaction intermediate.