Evidence for a polysaccharide-binding domain in Horrnoconis resinae glucoamylase P: effects of i ts proteolytic removal on substrate specificity and inhibition by P-cyclodextrin

Helsinki, Finland Ltd, POB 350, SF-00101 The hydrolysis of soluble starch, raw starch and pullulan with recombinant glucoamylase P from Hormoconis resinae was competitively inhibited by /?cyclodextrin with apparent Ki values of 190 pM, 13 pM and 1.4 pM, respectively. Inhibition of dextran hydrolysis was partial: a maximum inhibition of 22% was achieved with a dextran concentration of 0 3 x K, and up to 4 mM /?-cyclodextrin. Hydrolysis of short oligosaccharides was not inhibited by /?-cyclodextrin at levels up to 20 mM. The enzyme bound to raw starch at pH 4 3 and 4 "C with an association constant of 3.4 x l o 5 M-'. 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 K, 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 /?-cyclodextrin.