Residues essential for catalytic activity of soybean beta-amylase.
To determine which amino acid residues are essential for the catalytic activity of soybean beta-amylase, deoxyoligonucleotide site-directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in beta-amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue. Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean beta-amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non-mutant level, implying that this residue may also play a crucial role in beta-amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50 degrees C to 30 degrees C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme.
Totsuka
A
A
Genetic Engineering Laboratory, National Food Research Institute, Ibaraki, Japan.
Nong
V H
VH
Kadokawa
H
H
Kim
C S
CS
Itoh
Y
Y
Fukazawa
C
C
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013025:Glycine max / Q000201:enzymology*; D013696:Temperature; D001614:beta-Amylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005973:Glutamine; D001224:Aspartic Acid; D001614:beta-Amylase; D003545:Cysteine