Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum

• Published in: The Journal of biological chemistry Vol. 266; no. 16; pp. 10313 - 10318
• Main Authors: Tomme, P, Chauvaux, S, Beguin, P, Millet, J, Aubert, J.P, Claeyssens, M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05-06-1991
• Subjects: Amino Acid Sequence; Binding Sites; Blotting, Western; cellulase; Cellulase - antagonists & inhibitors; Cellulase - genetics; Cellulase - metabolism; cellulases; Chromatography, High Pressure Liquid; Clostridium - enzymology; Clostridium thermocellum; enzyme activity; Exact sciences and technology; glucans; histidine; Histidine - chemistry; Histidine - genetics; Kinetics; Mathematical analysis; Mathematics; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Potential theory; Sciences and techniques of general use; Sequence Homology, Nucleic Acid; site-directed mutagenesis; Enzyme; Active site; Site directed mutagenesis; Clostridiales; Inactivation; Chemical modification; Histidine; Enzymatic activity; Structure activity relation; Cellulase; Clostridium thermocellum; Clostridiaceae; Bacteria; Kinetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)99227-6

Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the k(cat) and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mm methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme.