Effect of an E461G mutation of beta-galactosidase (Escherichia coli, lac Z) on pL rate profiles and solvent deuterium isotope effects.

An E461G mutation of beta-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 beta-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 beta-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 beta-D-galactopyranosyl group from beta-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 beta-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 beta-D-galactopyranosyl reaction intermediate.