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

Authors
  • Tomme, P
  • Chauvaux, S
  • Beguin, P
  • Millet, J
  • Aubert, JP
  • Claeyssens, Marc
Publication Date
Jan 01, 1991
Source
Ghent University Institutional Archive
Keywords
Language
English
License
Unknown
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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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