Type CBM A Function

Although one can have a broad picture of how Type A function comes about, the detailed kinetics and thermodynamics still present unsolved problems. The planar, hydrophobic, aromatic face presented by all the protein folds that have Type A function suggests that the driving force is hydrophobic and that therefore Type A CBDs should bind preferentially to hydrophobic faces of the polysaccharide crystal. Indeed, with gold-labelled CBM 1 and CBM 3 binding to cellulose lo, (triclinic) crystals of Valonia ventricosa, electron microscopy reveals just that - preferential binding to the (110) face of the crystal. 

In perfect crystals these faces are fairly small - the major exposed faces along the fibre crystal are 100 and 010, which are hydrophilic - and are not large enough to account for the amount of CBM adsorbed. However, cellulose crystals will be damaged and this damage will be associated with removal of glucan chains from the corners of the crystal, increasing the effective hydrophobic area. 

However, the detailed kinetics and thermodynamics of Type A function present an as yet unsolved problem. With many CBMs, it is possible to obtain apparently conventional binding isotherms against various types of cellulose in the binding direction the cellulose does not bind more CBM if more is added and the amount of cellulose bound is constant with respect to time. However, if the solution is now diluted with buffer, no loss of CBM from the cellulose is seen, and if bound CBM is centrifuged off and placed in fresh buffer, no CBM is liberated the CBM binding is apparently irreversible. 

The binding does not appear irreversible in two dimensions, however two-dimensional diffusion on the surface of a Valonia ventricosa crystal to two CBM 2as from C.fimi was demonstrated. Obviously, having a CBM which bound a catalytic domain not only irreversibly but also immovably would vitiate its biological function. 

Making a biomolecular reaction unimolecular confers a maximum entropic advantage of 10 M. In an attempt to exploit this, a genetically engineered bivalent Type A CBM was constructed from the CBHII and CBHI CBDs from T. reesei just discussed. For a thermodynamically well-behaved system, the lower limit on the dissociation constant (M level) of the dimer from cellulose is therefore lQ KiK2, where Ki and K2 are the dissociation constants of the individual monomeric CBMs. The CBHII CBD of this system is not thermodynamically well behaved, but dissociation constants of the monomers are in the pM region and of the dimer only 10-15 times lower.  

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