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

P Tomme, S Chauvaux, P Beguin, J Millet, JP Aubert and Marc Claeyssens (1991) JOURNAL OF BIOLOGICAL CHEMISTRY. 266(16). p.10313-10318
abstract
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 K(m) 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.
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author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
SCHIZOPHYLLUM-COMMUNE, NUCLEOTIDE-SEQUENCE, CELLULOSE DEGRADATION, TRICHODERMA-REESEI, ESCHERICHIA-COLI, PROTEINS, GENE, DNA, CRYSTALLIZATION, INHIBITORS
journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
J. Biol. Chem.
volume
266
issue
16
pages
10313 - 10318
Web of Science type
Article
ISSN
0021-9258
language
English
UGent publication?
yes
classification
A1
id
222108
handle
http://hdl.handle.net/1854/LU-222108
date created
2004-05-24 13:24:00
date last changed
2016-12-19 15:38:50
@article{222108,
  abstract     = {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 K(m) 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.},
  author       = {Tomme, P and Chauvaux, S and Beguin, P and Millet, J and Aubert, JP and Claeyssens, Marc},
  issn         = {0021-9258},
  journal      = {JOURNAL OF BIOLOGICAL CHEMISTRY},
  keyword      = {SCHIZOPHYLLUM-COMMUNE,NUCLEOTIDE-SEQUENCE,CELLULOSE DEGRADATION,TRICHODERMA-REESEI,ESCHERICHIA-COLI,PROTEINS,GENE,DNA,CRYSTALLIZATION,INHIBITORS},
  language     = {eng},
  number       = {16},
  pages        = {10313--10318},
  title        = {Identification of a histidyl residue in the active-center of endoglucanase-D from Clostridium-ihermocellum},
  volume       = {266},
  year         = {1991},
}

Chicago
Tomme, P, S Chauvaux, P Beguin, J Millet, JP Aubert, and Marc Claeyssens. 1991. “Identification of a Histidyl Residue in the Active-center of endoglucanase-D from Clostridium-ihermocellum.” Journal of Biological Chemistry 266 (16): 10313–10318.
APA
Tomme, P., Chauvaux, S., Beguin, P., Millet, J., Aubert, J., & Claeyssens, M. (1991). Identification of a histidyl residue in the active-center of endoglucanase-D from Clostridium-ihermocellum. JOURNAL OF BIOLOGICAL CHEMISTRY, 266(16), 10313–10318.
Vancouver
1.
Tomme P, Chauvaux S, Beguin P, Millet J, Aubert J, Claeyssens M. Identification of a histidyl residue in the active-center of endoglucanase-D from Clostridium-ihermocellum. JOURNAL OF BIOLOGICAL CHEMISTRY. 1991;266(16):10313–8.
MLA
Tomme, P, S Chauvaux, P Beguin, et al. “Identification of a Histidyl Residue in the Active-center of endoglucanase-D from Clostridium-ihermocellum.” JOURNAL OF BIOLOGICAL CHEMISTRY 266.16 (1991): 10313–10318. Print.