Effect of the weak Ca(2+)-binding site of subtilisin J by site-directed mutagenesis on heat stability.
The functional role of the negatively charged amino acid residue in subtilisin J from Bacillus stearothermophilus has been investigated by site-directed mutagenesis. Glu-195 located at the weak Ca2+-binding site was replaced with Gln to examine the role of Glu-195 in the heat stability of subtilisin J. Mutant enzyme was expressed in Bacillus subtilis and was purified from the culture supernatant. When the mutant enzyme was expressed at 37 degrees C in the presence of 2mM calcium chloride, the pattern of enzyme production was quite different from that of wild-type. The purified Gln-195 mutant enzyme was analyzed with respect to optimal temperature, optimal pH, and heat stability. The mutation was found to decrease the heat stability but not catalytic efficiency (kcat/Km) and optimal pH. These results demonstrate the important role of the negatively charged side chains at the weak Ca(2+)-binding site in the heat stability of subtilisin.
Jang
J S
JS
Department of Life Science, Korea Advanced Institute of Science and Technology, Taejon.
Bae
K H
KH
Byun
S M
SM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004795:Enzyme Stability; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D005971:Glutamates; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C077349:subtilisin J; D018698:Glutamic Acid; D012697:Serine Endopeptidases; D013381:Subtilisins; D002118:Calcium