Site-directed mutagenesis of the putative active site of endoglucanase K from Bacillus sp. KSM-330.
The roles of one Glu and four Asp residues of endoglucanase K from Bacillus sp. KSM-330, which are conserved in all the endo-beta-glucanases in the family D, were analyzed by site-directed mutagenesis. The gene for endoglucanase K was mutated to replace Asp-154, Asp-191, Asp-193 or Asp-300 by Asn, or to replace Glu-130 by Gln in the encoded enzyme. Mutant and wild-type genes were separately expressed in Bacillus subtilis and the resultant enzymes were purified from the culture broth. All mutant enzymes exhibited the same mobility on SDS-polyacrylamide gel electrophoresis as the wild-type enzyme and gave similar circular dichroism spectra to that of the wild-type enzyme. Substitution of Glu-130, Asp-191, Asp-193 or Asp-300 significantly decreased the specific activity of the enzyme toward CM-cellulose. Kinetic analysis of the abilities of these mutant enzymes to liberate p-nitrophenol from p-nitrophenylcellotrioside revealed that all the mutant enzymes had very much lower kcat values than that of the wild-type enzyme, while the Km values of these mutant enzymes were almost the same as that of the wild-type enzyme. Of these Glu and Asp residues, Glu-130 and Asp-191 seem to be most likely to be catalytic residues because substitutions of these residues resulted in the lowest kcat values of the mutant enzymes.
Ozaki
K
K
Tochigi Research Laboratories, Kao Corporation, Tochigi, Japan.
Sumitomo
N
N
Hayashi
Y
Y
Kawai
S
S
Ito
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013696:Temperature
C089185:endoglucanase K; D002480:Cellulase