Active-site binding of glycosides by Thermomonospora fusca endocellulase E2.
The determination of the high-resolution structure of the Thermomonospora fusca endocellulase E2 catalytic domain makes it ideal for exploring cellulase structure-function relationships. Here we present binding parameters (Kd, DeltaH degrees, and DeltaS 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 glycosides 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 indicated 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 volume of the subsite -1 binding pocket is crucial for enzyme function. Similarly, MUXylGlc (with its xylosyl unit located in position -1) bound with 100-fold higher affinity than MU(Glc)2. These results are similar to those for the related Trichoderma reesei exocellulase CBH II. The binding data were compared with that previously reported for CBH II and interpreted in terms of the functional differences between endo- and exocellulases.
Barr
B K
BK
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA. bbarr@loyla.edu
Wolfgang
D E
DE
Piens
K
K
Claeyssens
M
M
Wilson
D B
DB
D000192:Actinomycetales / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D002475:Cellobiose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D012694:Serine / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D017186:Titrimetry; D014443:Tyrosine / Q000235:genetics
D001426:Bacterial Proteins; D005960:Glucosides; D006027:Glycosides; D008024:Ligands; D002475:Cellobiose; C014110:4-methylumbelliferyl glucoside; D006923:Hymecromone; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; C068475:4-methylumbelliferylcellobioside; D002480:Cellulase; D005947:Glucose