The catalytic consequences of experimental evolution. Transition-state structure during catalysis by the evolved beta-galactosidases of Escherichia coli (ebg enzymes) changed by a single mutational event.

1. The first chemical step in the hydrolysis of galactosylpyridinium ions by the evolvant ebg enzyme is less sensitive to leaving-group acidity than in the case of the wild-type ebg enzyme, implying less glycone-aglycone-bond fission at the transition state. 2. The first chemical step in the hydrolysis of aryl galactosides by ebg enzyme is probably less sensitive to leaving-group acidity than in the case of ebg enzyme, possibly as a consequence of resulting in more effective proton donation to the leaving aglycone. 3. alpha-Deuterium kinetic isotope effects of 1.1(0) and beta-deuterium kinetic isotope effects of 1.0(0) were measured for the hydrolysis of galactosyl-enzyme intermediates derived from ebg and ebg enzymes: these effects are not compatible with reaction of the sugar ring through a 4C1-like conformation, or with an ionic glycosyl-enzyme intermediate. 4. The variation with pH of steady-state kinetic parameters for hydrolysis of p-nitrophenyl galactoside by ebg and ebg enzymes and of 3-methylphenyl beta-galactoside, 3,4-dinitrophenyl beta-galactoside and beta-galactosyl-3-bromopyridinium ion by ebg enzyme was measured. The steep, non-classical, fall in activity against p-nitrophenyl galactoside at low pH observed with ebg and ebg enzymes is not observed with ebg enzymes.