Mutational analysis of the role of His452 of Saccharopolyspora rectivirgula β-galactosidase

To examine the role of His452 of the Saccharopolyspora rectivirgula β-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 β-galactosidase.