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 res
idues 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 a
mino acids, respectively, led to the complete elimination of activity,
but did not induce any significant changes in circular dichroic spect
ra or the binding affinity for cyclomaltohexaose, a substrate analogue
. Taking account of the results obtained here, the above two amino aci
d residues are involved in the catalytic site of soybean beta-amylase.
The replacement of glutamic acid at position 345 decreased activity t
o below 6% of the non-mutant level, implying that this residue may als
o play a crucial role in beta-amylase activity, although it may not be
involved at the catalytic site itself. In contrast, substitution of c
ysteinyl 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 therm
al stability of the enzyme.