title: Mutational analysis of the role of His452 of Saccharopolyspora rectivirgula beta-galactosidase.
abstract: To examine the role of His452 of the Saccharopolyspora rectivirgula beta-galactosidase in the binding of a tightly bound, catalytically important Mn2+ (i.e., class II Mn2+) ion, His452 was replaced with Phe or Glu and the respective site-directed mutants, H452F and H452E, were characterized. Neither mutant contained Mn2+ in an Mn2+-free buffer and both were virtually inactive in the absence of Mn2+ (their relative activities being less than 0.03% that of the fully activated wild-type enzyme). When Mn2+ was added, however, the mutants were activated to 3% (for H452F) and 0.8% (for H452E) of the full activity of the wild type. The Mn2+ concentrations needed for half-maximal activation of H452F and H452E were, respectively, 15,000 and 5000 times higher than the reported dissociation constant (2 nM) of the class II Mn2+, suggesting that His452 plays a key role in the binding of this catalytically important Mn2+. Activation of the mutants by Mn2+, albeit very weak, contrasts with a lack of any such metal activation previously observed with the two corresponding mutants of Escherichia coli lacZ beta-galactosidase.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 07 Aoba-yama, Sendai 980-8579, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Nakayama
authors.forename: Y
authors.initials: Y
authors.lastname: Goto
authors.forename: S
authors.initials: S
authors.lastname: Hasegawa
authors.forename: M
authors.initials: M
authors.lastname: Inohara-Ochiai
authors.forename: Y
authors.initials: Y
authors.lastname: Shibano
authors.forename: T
authors.initials: T
authors.lastname: Ashikari
authors.forename: T
authors.initials: T
authors.lastname: Nishino