title: Action patterns and mapping of the substrate-binding regions of endo-(1-->5)-alpha-L-arabinanases from Aspergillus niger and Aspergillus aculeatus.
abstract: The substrate binding sites of endo-(1-->5)-alpha-L-arabinanases (EC 3.2.1.99) from Aspergillus niger and Aspergillus aculeatus were investigated using reduced and regular (1-->5)-alpha-L-arabino-oligosaccharides and high performance anion exchange chromatographic analysis. Calculation of bond cleavage frequencies and kcat/K(m) parameters for these substrates enabled the determination of the number of arabinofuranosyl binding subsites and the estimation of the binding affinities of each subsite. The A. aculeatus endo-arabinanase has six subsites arranged symmetrically around the catalytic site, while the A. niger endo-arabinanase has five subsites; two from the catalytic site towards the non-reducing end of the bound substrate and three toward the reducing end. The two subsites directly adjacent to the catalytic sites in both the A. niger and A. aculeatus endo-arabinanase have near-zero net free energy of binding. These results are unlike most glycopyranosyl endo-hydrolases studied which have net negative (unfavourable) energies of interaction at these two subsites, and may be related to the greater conformational flexibility of arabinofuranosyl residues than glycopyranosyl residues. The complete subsite maps are also rationalized with regard to the observed action patterns of these enzymes on linear (1-->5)-alpha-L-arabinan.
authors.affiliation: Department of Food Science, Wageningen Agricultural University, The Netherlands.
authors.forename: S M
authors.initials: SM
authors.lastname: Pitson
authors.forename: A G
authors.initials: AG
authors.lastname: Voragen
authors.forename: J P
authors.initials: JP
authors.lastname: Vincken
authors.forename: G
authors.initials: G
authors.lastname: Beldman
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D001234:Aspergillus niger / Q000201:enzymology; D001665:Binding Sites; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D011134:Polysaccharides; C030080:araban; D006026:Glycoside Hydrolases; C082526:arabinan endo-1,5-alpha-L-arabinosidase