SUBSTITUTIONS FOR GLU-537 OF BETA-GALACTOSIDASE FROM ESCHERICHIA-COLI CAUSE LARGE DECREASES IN CATALYTIC ACTIVITY

Glu-537 of beta-galactosidase (EC 3.2.1.23) was replaced by Asp, Gln and Val using synthetic oligonucleotides. The k(cat.) values of the purified enzyme mixtures were reduced by about 100-fold for the Asp mutant, 30000-60000-fold for the Val mutant and 160000-300000-fold for the Gln mutant. The greatest differences in properties from the wild-type enzyme were found for the Asp-substituted enzyme: the K-m values increased (from 0.12 to 0.42 mM for o-nitrophenyl beta-D-galactopyranoside and from 0.04 to 0.37 mM for p-nitrophenyl beta-D-galactopyrano side), the K-i value for isopropyl beta-D-galactopyranoside increased (from 0.11 to 0.30 mM), the stability to heat decreased and methanol did not act as an acceptor. The enzymes with the other two substitutions had properties similar to those of the wild-type. For all three substituted enzymes, the inhibitory effects of the transition-state analogues (2-deoxy-2-amino-D-galactose and L-ribose) and the Mg2+ effects were similar to those of the normal enzyme. As all of the properties (except the k(cat.) values) of the Gln- and Val-substituted enzyme preparations were similar to those of the wild-type enzyme, the activities in those preparations were probably due to the presence of a few wild-type enzyme molecules (formed from misreads) among the substituted enzymes. The enzymes with Gin and Val substitutions appear to be totally inactive. The results obtained support a recent suggestion that Glu-537 is an important catalytic residue of beta-galactosidase.