title: Function of conserved tryptophans in the Aspergillus niger glucoamylase 1 starch binding domain.
abstract: Nuclear magnetic resonance (NMR) and ultraviolet (UV) difference spectroscopy were used to assess the role of a number of tryptophan residues in the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger. Wild-type SBD and three variant (W563K, W590K, and W615K) proteins were produced using an A. niger expression system. Titration studies were conducted with beta-cyclodextrin (betaCD), a cyclic analogue of starch, as the ligand. The NMR studies show that the W563K and W590K variants only bind 1 equiv while the wild-type protein forms a 2:1 (ligand:protein) complex. It also clearly demonstrates the abolition of binding at site 1 and site 2 in W590K and W563K, respectively. UV difference spectroscopy was used to calculate dissociation constants with addition of betaCD: 14.4 microM (apparent) for the wild type, 28.0 microM for W563K, and 6.4 microM for W590K. The implication of this is that the two binding sites have unequal contributions to the overall binding of the SBD which may be related to functional differences between the two binding sites. The low stability of the third variant, W615K, suggests that this tryptophan is not involved in binding but has an essential structural role.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
authors.forename: M P
authors.initials: MP
authors.lastname: Williamson
authors.forename: M F
authors.initials: MF
authors.lastname: Le Gal-Coëffet
authors.forename: K
authors.initials: K
authors.lastname: Sorimachi
authors.forename: C S
authors.initials: CS
authors.lastname: Furniss
authors.forename: D B
authors.initials: DB
authors.lastname: Archer
authors.forename: G
authors.initials: G
authors.lastname: Williamson
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D003505:Cyclodextrins / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D013056:Spectrophotometry, Ultraviolet; D013213:Starch / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014364:Tryptophan; D047392:beta-Cyclodextrins
chemical_list: D003505:Cyclodextrins; D047392:beta-Cyclodextrins; D014364:Tryptophan; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase; C031215:betadex