Catalytic mechanism of beta-amylase from Bacillus cereus var. mycoides: chemical rescue of hydrolytic activity for a catalytic site mutant (Glu367-->Ala) by azide.
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.
Miyake
Hideo
H
Laboratory of Enzyme Chemistry, Graduate School of Agriculture and Biological Science, Osaka Prefecture University, Sakai, 599-8531, Japan.
Otsuka
Chikako
C
Nishimura
Shigenori
S
Nitta
Yasunori
Y
D000409:Alanine / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008320:Maltose / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013696:Temperature; D001614:beta-Amylase / Q000737:chemistry*
D001386:Azides; D009844:Oligosaccharides; C019193:maltopentaose; D018698:Glutamic Acid; D008320:Maltose; D001614:beta-Amylase; D000409:Alanine