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.
Richard
J P
JP
Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, USA. jrichard@chem.buffalo.edu
Huber
R E
RE
McCall
D A
DA
D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005690:Galactose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D007763:Lac Operon / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D012997:Solvents; D001616:beta-Galactosidase / Q000235:genetics*
D007202:Indicators and Reagents; D012997:Solvents; D003903:Deuterium; D001616:beta-Galactosidase; D005690:Galactose