Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum.
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 kcat 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.
Tomme
P
P
Laboratorium voor Biochemie, Rijksuniversiteit Gent, K. L. Ledeganckstraat, Belgium.
Chauvaux
S
S
Béguin
P
P
Millet
J
J
Aubert
J P
JP
Claeyssens
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012689:Sequence Homology, Nucleic Acid
D006639:Histidine; D002480:Cellulase