Site-directed mutagenesis of conserved residues of Clostridium thermocellum endoglucanase CelC.
Four conserved residues of Clostridium thermocellum endoglucanase CelC were replaced by site-directed mutagenesis. Proteins mutated in His-90, Asn-139 and Glu-140 showed strongly reduced activity, in agreement with predictions of sequence alignments. Mutations in Glu-140 did not result in any detectable change in Km, or apparent size, suggesting that Glu-140 is directly involved in catalysis. The pH optimum of the proteins carrying the Glu-140/Ala and Glu140/Gln mutations was lower than that of the wild type, whereas the activity vs. pH profile of Glu-140/Asp CelC was similar to that of the wild type, suggesting that Glu-140 may act as a proton donor.
Navas
J
J
Département des Biotechnologies, Institut Pasteur, Paris, France.
Béguin
P
P
D000595:Amino Acid Sequence; D001120:Arginine; D001216:Asparagine; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D005971:Glutamates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; D001216:Asparagine; D001120:Arginine; C078899:endoglucanase CelC; D002480:Cellulase