Specificity and affinity of substrate binding by a family 17 carbohydrate-binding module from Clostridium cellulovorans cellulase 5A.
The C-terminal carbohydrate-binding module (CBM17) from Clostridium cellulovorans cellulase 5A is a beta-1,4-glucan binding module with a preference for soluble chains. CBM17 binds to phosphoric acid swollen Avicel (PASA) and Avicel with association constants of 2.9 (+/-0.2) x 10(5) and 1.6 (+/-0.2) x 10(5) M(-1), respectively. The capacity values for PASA and Avicel were 11.9 and 0.4 micromol/g of cellulose, respectively. CBM17 did not bind to crystalline cellulose. CBM17 bound tightly to soluble barley beta-glucan and the derivatized celluloses HEC, EHEC, and CMC. The association constants for binding to barley beta-glucan, HEC, and EHEC were approximately 2.0 x 10(5) M(-1). Significant binding affinities were found for cello-oligosaccharides greater than three glucose units in length. The affinities for cellotriose, cellotetraose, cellopentaose, and cellohexaose were 1.2 (+/-0.3) x 10(3), 4.3 (+/-0.4) x 10(3), 3.8 (+/-0.1) x 10(4), and 1.5 (+/-0.0) x 10(5) M(-1), respectively. Fluorescence quenching studies and N-bromosuccinimide modification indicate the participation of tryptophan residues in ligand binding. The possible architecture of the ligand-binding site is discussed in terms of its binding specificity, affinity, and the participation of tryptophan residues.
Boraston
A B
AB
Protein Engineering Network of Centres of Excellence, Department of Microbiology and Immunology, and The Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, Canada.
Chiu
P
P
Warren
R A
RA
Kilburn
D G
DG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry; D002480:Cellulase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D005936:Glucans / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005936:Glucans; D009844:Oligosaccharides; D010446:Peptide Fragments; D011134:Polysaccharides; C074074:cellulose binding protein A, Clostridium cellulovorans; C099423:cellohexaose; C019193:maltopentaose; D012965:Sodium Chloride; C040088:1,4-glucan; C407024:endoglucanase Cel5A; D002480:Cellulase