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, initrophenyl -2-deoxy-2-fluoro-beta-D-cellobioside, was synthesized and used to tra p the intermediate formed during catalysis by endoglucanase C (CelC) f rom Clostridium thermocellum. Ion spray mass spectrometry confirmed th e 1:1 stoichiometry of the incorporation of the inactivator into the e nzyme. Inactivation followed the required pseudo first-order kinetic b ehavior and kinetic parameters for the process were determined. Althou gh the intermediate trapped was relatively stable (t1/2 = 25 h), turno ver was facilitated by transglycosylation following the addition of ph enyl-beta-D-thiocellobioside and cellobiose, thus demonstrating the ca talytic 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 suga r to the peptide. Alignment of all known related beta-1,4-glucanases s howed that Glu280 is strictly conserved in family A enzymes, consisten t with its key role in catalysis.