ACTIVE-SITE BINDING OF GLYCOSIDES BY THERMOMONOSPORA-FUSCA ENDOCELLULASE E2

The determination of the high-resolution structure of the Thermomonosp ora fusca endocellulase E2 catalytic domain makes it ideal for explori ng cellulase structure-function relationships. Here we present binding parameters (K-d, Delta H degrees, and Delta S degrees) describing the interaction of E2 with 4-methylumbelliferyl glycosides, determined by titrating the quenching of ligand fluorescence in equilibrium binding experiments, Quenched MU(Glc)(2)/E2 complexes were used as indicators in displacement titrations to measure the binding of natural glycosid es and also of a nonhydrolyzable cellotetraose analogue. Binding of MU (Glc)(2) and cellotriose were also determined by titration calorimetry , The results show that E2 binds glycosides exclusively in its active- site cleft, with high affinity and specificity. The observed patterns of ligand hydrolysis and the results with MU(Glc)2 as a substrate indi cated that ligands bound to E2 with their nonreducing ends in position -2, consistent with the position of cellobiose in the E2cd structure. Polymerase chain reaction (PCR) mutagenesis of the conserved residue Tyr 73 (in E2 binding subsite -1) to Phe and Ser produced enzymes with lower activity but higher binding affinities, indicating that the vol ume of the subsite -1 binding pocket is crucial for enzyme function. S imilarly, MUXylGlc (with its xylosyl unit located in position -1) boun d with 100-fold higher affinity than MU(Glc)2. These results are simil ar to those for the related Trichoderma reesei exocellulase CBH II, Th e binding data were compared with that previously reported for CBH II and interpreted in terms of the functional differences between endo- a nd exocellulases.