The classification above resolves whether catalytic domains of cellulases hydrolyze glycosidic bonds at the end of cellulose chains (exocellulases) or in the middle (endocellulases). An important additional distinction among endocellulases is whether they are processive (binding to a cellulase chain and then hydrolyzing at multiple sites, perhaps in sequence on the chain) or nonprocessive (binding, hydro­lyzing a single bond, and releasing). Comparison of multiple crystal structures of cellulases has revealed some of the basis of processivity, along with the basis for endo — vs. exocellulolytic activity (see [84] for a review). It should be emphasized that the type of processivity discussed above relates to individual catalytic domains, since the attachment of CBDs in cis is likely to significantly enhance processivity by tethering the enzyme to the substrate, as is the presence of multiple catalytic domains in a single polypeptide chain.

An important subdivision of the exocellulases is whether they attack the reduc­ing end or nonreducing end of the cellulose chain [2]. In many cases, exocellulases produce the disaccharide cellobiose and are appropriately designated “cellobiohy — drolases,” although many exocellulases also produce longer oligosaccharides such as cellotriose and cellotetraose.

A final mechanistic subdivision that can be made is whether a cellulase cleaves the b-glucosidic bond with retention of chirality at the C1 position (producing the b-anomer) or with inversion (producing the a-anomer). For both mechanisms, cel — lulases use a pair of acidic (asp/glu) residues in a general acid/general base scheme. Other common features include water attack at the C1 position on the nonreducing side of the glycosidic bond to be cleaved and displacement of the O4-sugar. In the inverting mechanism, the water attacks C1 directly, with one of the acidic residues acting as a general base to abstract a proton from the attacking water, and the other acting as a general acid to protonate the O4 leaving group. In the retaining mecha­nism, the general base forms a covalent adduct (an ester linkage between the side — chain carboxyl and C1), displacing the O4, with protonation from the general acid. In a second step, water is activated by the general acid (now in its deprotonated conjugate base form), which attacks the C1 and displaces the C1-asp/glu linkage.