Catalytic consequences of experimental evolution. Part 3. Construction of reaction profiles for hydrolysis of lactose by ebg°, ebga, and ebgb enzymes via measurement of the enzyme-catalysed exchange of D-[1-18O]galactose by 13C nuclear magnetic resonance spectroscopy

Abstract

Rate-constants at 37°C for the exchange of D-[1-¹⁸O]galactose with solvent, catalysed by the wild-type second β-galactosidase of E. coli, ebg°, and the experimental evolvants ebg^a and ebg^b have been measured. These have been used with other data to construct substantially complete free-energy profiles for the wild-type enzyme and the two evolvants selected for catalytic efficiency on lactose. The consequences of the ebg°→ebg^a change are not simple, but the ebg°→ebg^b change can be regarded as largely a stabilisation of the galactosyl-enzyme intermediate, considered to be the second most likely evolutionary change in a free-energy profile by W. J. Albery and J. R. Knowles, Biochemistry, 1976, 15, 5631.