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

Toru Nakayama, Yuka Goto, Satoshi Hasegawa, Misa Inohara-Ochiai, Yuji Shibano, Toshihiko Ashikari, Tokuzo Nishino

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)535-539
Number of pages5
JournalJournal of Bioscience and Bioengineering
Volume90
Issue number5
DOIs
Publication statusPublished - 2000 Jan 1

Keywords

  • Metal ion requirements
  • Saccharopolyspora rectivirgula
  • Site-directed mutagenesis
  • β-galactosidase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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