Differences in the effects of pH on the hydrolytic and transgalactosylic reactions of β-galactosidase (Escherichia coli)

Steady-state kinetic studies with β-galactosidase and various substrates were carried out to determine why the ratio of transgalactosylis to hydrolysis increased as a function of pH from 7.0 to 10.0. The rate constant [Formula: see text] for the formation of galactose (hydrolytic reaction) decreased whereas the rate constant (k4) for the transgalactosylic reaction (i.e., the formation of allolactose) remained constant. The equilibrium constant for acceptor dissociation from the galactosyl form of the enzyme was also unaffected by pH in the range studied; this was true whether the acceptor was glucose, sucrose, or glycerol. These results suggest that there is a group of high pKa at, or of influence at, the enzyme's active site which affects hydrolysis but not transgalactosylis. A further finding was that the rate constant for the breakage of the glycosidic bond decreased with pH in a manner different from the change observed for the hydrolytic rate constant (pKa 9.4 for glycosidic breakage as compared with 8.6 for hydrolysis). This could explain why the pH optimum for (β-galactosidase activity varies with substrate; different steps are rate limiting for different substrates.