Catalytic mechanism of beta-amylase from Bacillus cereus var. mycoides: chemical rescue of hydrolytic activity for a catalytic site mutant (Glu367-->Ala) by azide |
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Authors: | Miyake Hideo Otsuka Chikako Nishimura Shigenori Nitta Yasunori |
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Affiliation: | Laboratory of Enzyme Chemistry, Graduate School of Agriculture and Biological Science, Osaka Prefecture University, Sakai, 599-8531, Japan. |
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Abstract: | The hydrolytic activity of beta-amylase from Bacillus cereus var. mycoides was lost on replacement of either of the catalytic residues (Glu172 or Glu367) with an alanyl residue. When maltopentaose and 2 M azide existed together mutant, E367A cleaved the glucosidic linkage of maltopentaose and produced maltose at pH 7.0 and 25 degrees C, but the other mutants (E172A and double mutant E172A/E367A) did not. This indicates that azide acts as a general base instead of E367 and Glu172 acting as general acids, and that the hydroxide ion generated from a water molecule activated by azide attacks a reactive pyranose nucleophilically so that beta-maltose is produced. |
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