GLU-416 of β-Galactosidase (Escherichia coli) Is a MG2+Ligand and β-Galactosidases with Substitutions for GLU-416 Are Inactivated, Rather than Activated, by MG2+

Abstract Glu-416 of β-galactosidase (E. coli) was replaced with Gln and Val using site-directed mutagensis. The substituted enzymes displayed a greatly decreased sensitivity to Mg2+. Equilibrium dialysis studies indicated that wild-type β-galactrosidase bound Mg2+tightly, whereas E416V-β-galactosidase did not. In addition, the pH profile of E416V-β-galactosidase was unaffected by the presence of or absence of 1 mM Mg2+. Surprisingly, both substituted enzymes were inactivated, rather than activated, by Mg2+but high amounts of Mg2+were needed (1mM). E416Q-β-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+.