TY - JOUR
T1 - Mutational analysis of the role of His452 of Saccharopolyspora rectivirgula β-galactosidase
AU - Nakayama, Toru
AU - Goto, Yuka
AU - Hasegawa, Satoshi
AU - Inohara-Ochiai, Misa
AU - Shibano, Yuji
AU - Ashikari, Toshihiko
AU - Nishino, Tokuzo
N1 - Funding Information:
We thank Mitsuru Sato, Tohoku University, for his technical assistance. This work was supported in part by a grant for the development of highly functional materials by structural modification of carbohydrates (Project on Glycotechnology) from the Ministry of Agriculture, Forestry, and Fisheries, Japan.
PY - 2000
Y1 - 2000
N2 - 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.
AB - 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.
KW - Metal ion requirements
KW - Saccharopolyspora rectivirgula
KW - Site-directed mutagenesis
KW - β-galactosidase
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U2 - 10.1016/S1389-1723(01)80036-0
DO - 10.1016/S1389-1723(01)80036-0
M3 - Article
C2 - 16232905
AN - SCOPUS:0034319165
VL - 90
SP - 535
EP - 539
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
SN - 1389-1723
IS - 5
ER -