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 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.