Evidence for a polysaccharide-binding domain in Hormoconis resinae glucoamylase P: effects of its proteolytic removal on substrate specificity and inhibition by beta-cyclodextrin.
The hydrolysis of soluble starch, raw starch and pullulan with recombinant glucoamylase P from Hormoconis resinae was competitively inhibited by beta-cyclodextrin with apparent Ki values of 190 microM, 13 microM and 1.4 microM, respectively. Inhibition of dextran hydrolysis was partial: a maximum inhibition of 22% was achieved with a dextran concentration of 0.3 x Km and up to 4 mM beta-cyclodextrin. Hydrolysis of short oligosaccharides was not inhibited by beta-cyclodextrin at levels up to 20 mM. The enzyme bound to raw starch at pH 4.3 and 4 degrees C with an association constant of 3.4 x 10(5) M-1. Sequence alignment studies showed raw-starch-binding consensus amino acids in the C-terminal part of glucoamylase P. Partial hydrolysis with papain resulted in degradation of deglycosylated glucoamylase P into three fragments of 53, 51 and 14 kDa, respectively, as estimated by SDS-PAGE. The amino-terminal sequences of the 51 and 53 kDa fragments were identical with that of native glucoamylase P. The amino terminus of the 14 kDa fragment (Ser-Ser-X-Gln-Val-Ser-), corresponded to the sequence starting at residue 474 of intact glucoamylase P. Kinetic measurements of truncated glucoamylase P showed changes in the Km values of larger polysaccharides, but no changes in kcat values compared to the intact enzyme. It was concluded that glucoamylase P contains a catalytic core domain and a raw-starch-binding domain involved in inhibition of polysaccharide hydrolysis by beta-cyclodextrin.
Fagerström
R
R
Research Laboratories Alko Ltd, Helsinki, Finland.
D000595:Amino Acid Sequence; D001665:Binding Sites; D002963:Cladosporium / Q000201:enzymology* / Q000235:genetics; D003505:Cyclodextrins / Q000494:pharmacology; D005656:Fungal Proteins; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011134:Polysaccharides; D013213:Starch; D013379:Substrate Specificity; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D005656:Fungal Proteins; D011134:Polysaccharides; D047392:beta-Cyclodextrins; D013213:Starch; C086694:GamP protein, Amorphotheca resinae; D005087:Glucan 1,4-alpha-Glucosidase; C031215:betadex