Glu-416 of beta-galactosidase (Escherichia coli) is a Mg2+ ligand and beta-galactosidases with substitutions for Glu-416 are inactivated, rather than activated, by MG2+.

Glu-416 of beta-galactosidase (E. coli) was replaced with Gln and Val using site-directed mutagenesis. The substituted enzymes displayed a greatly decreased sensitivity to Mg2+. Equilibrium dialysis studies indicated that wild-type beta-galactosidase bound Mg2+ tightly, whereas E416V-beta-galactosidase did not. In addition, the pH profile of E416V-beta-galactosidase was unaffected by the presence or absence of 1 mM Mg2+. Surprisingly, both substituted enzymes were inactivated, rather than activated, by Mg2+ but high amounts of Mg2+ were needed (1 mM). E416Q-beta-galactosidase was unstable when stored in the presence of Mg2+. The substituted enzymes displayed a dramatically lowered affinity for the synthetic substrate, ONPG, and for IPTG (a substrate analog inhibitor) in both the presence and the absence of Mg2+.