GLU-416 OF BETA-GALACTOSIDASE (ESCHERICHIA-COLI) IS A MG2-GALACTOSIDASES WITH SUBSTITUTIONS FOR GLU-416 ARE INACTIVATED, RATHER THAN ACTIVATED, BY MG2+( LIGAND AND BETA)

Glu-416 of beta-galactosidase (E. coli) was replaced with Gln and Val using site-directed mutagenesis. The substituted enzymes displayed a g reatly decreased sensitivity to Mg2+. Equilibrium dialysis studies ind icated that wild-type beta-galactosidase bound Mg2+ tightly, whereas E 416V-beta-galactosidase did not. In addition, the pH profile of E416V- beta-galactosidase was unaffected by the presence or absence of 1 mM M g2+, Surprisingly, both substituted enzymes were inactivated, rather t han activated, by Mg2+ but high amounts of Mg2+ were needed ( 1 mM). E 416Q-beta-Galactosidase was unstable when stored in the presence of Mg 2+. The substituted enzymes displayed a dramatically lowered affinity for the synthetic substrate, ONPG, and for IPTG (a substrate analog in hibitor) in both the presence and the absence of Mg2+. (C) 1996 Academ ic Press, Inc.