Site directed substitutions suggest that His-418 of beta-galactosidase (E. coli) is a ligand to Mg2+.
Site directed mutagenesis was used to replace His-418 of beta-galactosidase with Phe (H418F) or Glu (H418E). Kinetic analysis revealed that H418F beta-galactosidase was not significantly affected by the presence of Mg2+ whereas H418E beta-galactosidase retained its sensitivity to Mg2+. H418F had a kcat similar to that of Mg(2+)-free wild type beta-galactosidase. Its pH profile was shifted 1.0 pH unit lower on the alkaline side as compared to wild type beta-galactosidase (with Mg2+). This was similar to the shifting of the wild type beta-galactosidase pH profile when Mg2+ was absent. H418E beta-galactosidase was inactivated (rather than activated) by Mg2+ binding. Equilibrium dialysis studies indicated that H418E and wild type beta-galactosidase bind Mg2+ tightly whereas H418F does not. The results indicate that His-418 is probably a ligand to Mg2+.
Roth
N J
NJ
Division of Biochemistry, Faculty of Science, University of Calgary, Alberta.
Huber
R E
RE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D008024:Ligands; D011994:Recombinant Proteins; D006639:Histidine; D001616:beta-Galactosidase; D008274:Magnesium