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+.
Roth
N J
NJ
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Huber
R E
RE
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid; D005973:Glutamine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014633:Valine; D001616:beta-Galactosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D008024:Ligands; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D001616:beta-Galactosidase; D014633:Valine; D008274:Magnesium