Modification of catalytically important carboxy residues in endoglucanase D from Clostridium thermocellum.
Endoglucanase D (EC 3.2.1.4; EGD) from Clostridium thermocellum is rapidly (k = 216 M-1.min-1) and almost completely (greater than 95%) inactivated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulphonate). Spectrophotometric analysis at 340 nm reveals that eight carboxy residues react, whereas specific ligands protect one residue against modification. The enzyme retains it full activity under the latter conditions. The kinetics and pH-dependence of inactivation point towards the involvement of one or more essential carboxy groups with a pKa of 5.7-5.8. Samples modified in the absence or presence of ligand were analysed by reversed-phase liquid chromatography after proteolysis with subtilisin. Dual-wavelength monitoring at 214 and 340 nm during this fractionation leads to the identification of a putatively active-site peptide (Gly-508-Ala-562) which was further characterized by amino acid and partial N-terminal sequence analyses. Asp-546 and Glu-555 are postulated as possible active-site residues. This follows from alignments using ten endoglucanase sequences belonging to the same family. Strong local conservation suggests that this C-terminal sequence is structurally and/or functionally important.
Tomme
P
P
Laboratorium voor Biochemie, Rijksuniversiteit Gent, Belgium.
van Beeumen
J
J
Claeyssens
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002264:Carboxylic Acids / Q000378:metabolism; D002384:Catalysis; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016415:Sequence Alignment; D013379:Substrate Specificity
D000596:Amino Acids; D002264:Carboxylic Acids; D007202:Indicators and Reagents; D007555:Isoxazoles; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D002480:Cellulase