Specificity of the binding domain of glucoamylase 1.
Glucoamylase 1 from Aspergillus niger hydrolyses granular starch at an increased rate due to the presence of a C-terminal starch-binding domain. This domain was isolated and shown to bind to the malto-oligosaccharides Glc2 to Glc11 with a stoichiometry of 1 mol ligand/mol protein. The affinity for these ligands increased with increasing degree of polymerisation until Glc9, above which no further increase was observed. We suggest that this indicates that for maximum affinity the substrate should be able to form a helical conformation, which mimics the conformation of amylose in granular starch. We propose a model of how the complex between the malto-oligosaccharides and the binding domain is formed and indicate how this affects the differences in binding modes for soluble and insoluble substrates. Glucono-1,5-lactone interacts with the binding domain at a different site to the malto-oligosaccharides allowing the formation of a ternary complex between the binding domain, a malto-oligosaccharide and glucono-1,5-lactone. The binding domain also binds to linear alpha-1,6-linked glucose digosaccharides (dextran), but with much lower affinity than for alpha-1,4-linked glucose. This ligand appears to interact with the binding domain at both binding sites, i.e. at the site to which the malto-oligosaccharides bind and also at the site to which glucono-1,5-lactone binds. The relevance of the results to the mechanism of action of other polysaccharide-hydrolysing enzymes containing both a catalytic and a binding domain is discussed.
Belshaw
N J
NJ
Department of Food Molecular Biochemistry, AFRC Institute of Food Research, Norwich Laboratory, England.
Williamson
G
G
D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D001667:Binding, Competitive; D002236:Carbohydrate Conformation; D003911:Dextrans / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism*; D005942:Gluconates / Q000378:metabolism; D007783:Lactones; D008433:Mathematics; D008968:Molecular Conformation; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011108:Polymers; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D003911:Dextrans; D005942:Gluconates; D007783:Lactones; D009844:Oligosaccharides; D011108:Polymers; C021705:maltooligosaccharides; D005087:Glucan 1,4-alpha-Glucosidase; C010730:beta-glucono-1,5-lactone