The predominant molecular state of bound enzyme determines the strength and type of product inhibition in the hydrolysis of recalcitrant polysaccharides by processive enzymes [Enzymology]

March 12th, 2015 by Kuusk, S., Sorlie, M., Valȷamae, P.

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate limiting for these enzymes. Here, we performed a comparative study of two well-characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely, chitobiose for ChiA and cellobiose for Cel7A. The products behaved as non-competitive inhibitors according to studies using the 14C labeled crystalline polymeric substrates 14C-chitin nanowhiskers and 14C-bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki(obs) values were 0.45 ± 0.08 mM for ChiA and 0.17 ± 0.02 mM for Cel7A. However, in contrast to ChiA, the Ki(obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active site mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation.
  • Posted in Journal of Biological Chemistry, Publications
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