Use of isothermal titration calorimetry to characterize IPE variants of T. reesei Cel3A

The cellobiase activities of five T. reesei Cel3A mutants were assayed by isothermal titration calorimetry (MicroCal).  The five mutants were previously identified from primary screening of site-evaluation and combinatorial libraries, and selected based on their performance results on improved stability and activity over the wild type enzyme.  Analysis of the substrate saturation curves generated by ITC revealed that all mutants are subject to cellobiose inhibition at concentrations above 2.5mM.  In addition, all mutants, including the wild type, are best modeled using a catalytic inhibition mechanism that incorporates the reversible binding of a second cellobiose molecule to the enzyme-cellobiose complex intermediate (ES).  The resulting SES complex is capable of breaking down to the enzyme-substrate complex and product.  Using this mechanism for analysis revealed two Cel3A mutants with K_cat values of 1.6 and 2.0 times higher than the wild type parent.  The biophysical assay employed here offers many advantages over conventional colorimetric kinetic methods; key among them is the generation of real-time data, the authentication of the pseudo steady state assumption, and a high sensitivity for measuring reducing ends (~2-4ρmol of reducing end product per second).