On the basis of the structural similarity between chitosanase and hen egg white lysozyme (38),(41), six binding subsites, each of which can accommodate one D-glucosamine residue, were estimated by energy minimization. They involve the following amino acid residues:

* Tyr-34 is the best candidate for the interacting amino acid residue at subsite E. The presently observed distance between the -OH of Tyr-34 and the amino group of the sugar residue at subsite E is too long for an efficient interaction. The contact would become possible only if a change of enzyme conformation occured after substrate binding.

On the basis of the crystallographic analogy with hen egg white lysozyme, a model of chitosanase-hexaglucosamine interaction was proposed (38). This model adopted an asymmetrical localization of the hexasaccharide in the binding cleft of chitosanase, with subsites (-4)(-3)(-2)(-1)(+1)(+2). According to this model, the hexasaccharide should be cleaved preferably into a tetramer and a dimer. The tetramer could be cleaved again into two dimers.

However, a detailed study of the interaction between chitosanase and hexaglucosamine led to an alternative model. First, the main product resulting from hexaglucosamine hydrolysis turned out to be the trimer and not the dimer ((26). This was the first indication that the hexaglucosamine-chitosanase interaction is rather symmetrical. More recently (67), theoretical calculations were applied to hydrolysis patterns obtained with the wild type enzyme and two mutants at the Asp57 position, D57N and D57A (Asp 57 is one of the four carboxylic residues identified by crystallography as possible candidates for interaction with substrate). These studies allowed to conclude that: a) Asp57 is one of the most important substrate-interacting residues in chitosanase, b) Asp57 interacts with substrate at subsite (-2) and c) the productive binding of hexaglucosamine to the enzyme is better described by a symmetrical model including subsites (-3)(-2)(-1)(+1)(+2)(+3) with cleavage occuring in the middle. This model is thus analogous to models previously deduced for barley chitinase and goose egg white lysozyme (68).

Time-course of hexaglucosamine hydrolysis by wild-type chitosanase (26), (67)