| Abstract
| - The modes of action of three family 18 chitinases (ChiA, ChiB, and ChiC) from Serratiamarcescens during the degradation of a water-soluble polymeric substrate, chitosan, were investigatedusing a combination of viscosity measurements, reducing end assays, and characterization of the size-distribution of the oligomeric products. All three enzymes yielded a fast reduction in molecular weight ofthe chitosan substrate at a very early stage of hydrolysis, which is typical for endo-acting enzymes. ForChiA and ChiB, this is inconsistent with the previously proposed exo-attack mode of action. The maindifference between ChiA, ChiB, and ChiC is the degree of processivity. ChiC is an endo enzyme with noapparent processivity. ChiA and ChiB are processive enzymes in which the substrate remains bound tothe active cleft after successful hydrolysis and is moved along for the next hydrolysis to occur. ChiA andChiB perform on average 9.1 and 3.4 cleavages, respectively, for the formation of each enzyme−substratecomplex. ChiA and ChiB have deep, tunnel-like substrate-binding grooves. The demonstration of endoactivity shows that substrate binding must involve the temporary restructuring of the loops that make upthe roofs of the substrate-binding grooves, similar to what has been proposed for cellobiohydrolase Cel6A.The data suggest that the exo-type of activity observed for ChiA and ChiB during the degradation ofsolid crystalline chitin is due to the better accessibility of chain ends, rather than intrinsic enzyme properties.
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