GLU280 IS THE NUCLEOPHILE IN THE ACTIVE-SITE OF CLOSTRIDIUM-THERMOCELLUM CELC, A FAMILY-A ENDO-BETA-1,4-GLUCANASE

A new mechanism-based inactivator of beta-1,4-glucanases, 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-cellobioside, was synthesized and used to trap the intermediate formed during catalysis by endoglucanase C (CelC) from Clostridium thermocellum. Ion spray mass spectrometry confirmed the 1:1 stoichiometry of the incorporation of the inactivator into the enzyme. Inactivation followed the required pseudo first-order kinetic behavior and kinetic parameters for the process were determined. Although the intermediate trapped was relatively stable (t1/2 = 25 h), turnover was facilitated by transglycosylation following the addition of phenyl-beta-D-thiocellobioside and cellobiose, thus demonstrating the catalytic competence of the trapped intermediate. The nucleophilic amino acid residue involved was identified as Glu280 by labeling the enzyme with tritiated inactivator, cleaving it into peptides and sequencing the radiolabeled peptide. Ion spray mass spectrometric analysis of the peptide confirmed the sequence and the mode of attachment of the sugar to the peptide. Alignment of all known related beta-1,4-glucanases showed that Glu280 is strictly conserved in family A enzymes, consistent with its key role in catalysis.