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 k_cat/K_m for wild-type enzyme-catalyzed hydrolysis of 4-nitrophenyl β-D-galactopyranoside (Gal-OPNP) and a decrease from (k_cat)_HOH/(k_cat)_DOD = 1.7 to (k_cat)_HOH/(k_cat)_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 k_s 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.