Enzymes with Multiple Subsites such as Polysaccharidases

5 ENZYMES WITH MULTIPLE SUBSITES SUCH AS POLYSACCHARIDASES 

The commonest simple way to distinguish exo- from e fi o-acting enzymes is to measure the generation of reducing sugar equivalents and the reduction of substrate viscosity as the enzyme acts on a soluble polymer. The intrinsic viscosity of a linear polysaccharide is proportional to the molecular weight 

On the other hand, an exo-acting enzyme will bring about only a very marginal reduction in the DP of the polymer for each catalytic event. In tests of cellulases, it is usual to monitor both reducing sugar and viscosity as the enzyme in question hydrolyses carboxymethylated cellulose. 

The idea that polysaccharide-polysaccharidase interaction could be treated simply as the sum of the interactions of various subsites is intuitively attractive. Hiromi first tackled the algebra, but made the assumption, now known to be implausible, that the microscopic rate constant for bond cleavage in the ES complex was independent of subsite occupancy. This is very unlikely for enzymes in general terms, since it is now accepted that they exploit interactions with the substrate remote from the site of bond cleavage to lower the free energy for the catalysed reaction (the Circe effect, Section 5.4.5.3). The Hiromi assumption was shown to be incorrect by direct experiments with an endoxylanase. A better fit to experimental data was obtained if the subsite affinities were calculated for the first irreversible transition state i.e. on kcat/A)n and cleavage patterns of oligosaccharides), but careful analysis in some systems indicated that even this approximation failed.  

Processivity may be encountered with enzymes acting on polysaccharides, that is, the substrate remains bound to the enzyme for more than one catalytic event. In the case of enzymes acting on insoluble substrates, processivity is revealed by a high proportion of soluble to insoluble reducing sugar, as was the case with the GH 48 cellobiohydrolase of Clostridium thermocellum acting on 

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