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

An E46IG mutation of beta -galactosidase results in the disappearance of th e 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-galactopyra noside (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 G lu 461 is protonated at catalytically active free beta -galactosidase and t hey are consistent with a role for this residue in Bronsted 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 -galacto sidase to water cannot be simply explained by a mechanism in which the sing le side chain of Glu 461 functions to provide general acid catalysis in the rate limiting step for formation of the beta -D-galactopyranosyl intermedi ate and general base catalysis of breakdown of this intermediate. Evidence is presented that there may be different catalytic mechanisms, with differe nt roles for the side chain for Glu-461, for nucleophilic addition of water and of small alkyl alcohols to the beta -D-galactopyranosyl reaction inter mediate. (C) 2001 Academic Press.