SITE-DIRECTED SUBSTITUTIONS SUGGEST THAT HIS-418 OF BETA-GALACTOSIDASE (ESCHERICHIA-COLI) IS A LIGAND TO MG2+

Site directed mutagenesis was used to replace His-418 of beta-galactos idase with Phe (H418F) or Glu (H418E). Kinetic analysis revealed that H418F beta-galactosidase was nor significantly affected by the presenc e of Mg2+ whereas H418E beta-galactosidase retained its sensitivity to Mg2+. H418F had a k(cat) similar to that of Mg2+-free wild type beta- galactosidase. Its pH profile was shifted 1.0 pH unit lower on the alk aline side as compared to wild type beta-galactosidase (with Mg2+). Th is 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+ tight ly whereas H418F (C) 1994 Academic Press, Inc.