Mutational Analysis of the Role of His452 of Saccharopolyspora rectivirgula β-Galactosidase

概要

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To examine the role of His452 of the Saccharopolyspora rectivirgula β-galactosidase in the binding of a tightly bound, catalytically important Mn^<2+>(i.e., class II Mn^<2+>) ion, His452 was replaced with Phe or Glu and the respective site-directed mutants, H452F and H452E, were characterized. Neither mutant contained Mn^<2+> in an Mn^<2+>-free buffer and both were virtually inactive in the absence of Mn^<2+>(their relative activities being less than 0.03% that of the fully activated wild-type enzyme). When Mn^<2+> 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 Mn^<2+> 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 Mn^<2+> suggesting that His452 plays a key role in the binding of this catalytically important Mn^<2+>. Activation of the mutants by Mn^<2+> 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.
社団法人日本生物工学会の論文
2000-11-25