Identification of the Catalytic Residues of Bifunctional Glycogen Debranching Enzyme

Abstract

Eukaryotic glycogen debranching enzyme (GDE) possesses two different catalytic activities (oligo-1,431,4-glucantransferase/amylo-1,6-glucosidase) on a single polypeptide chain. To elucidate the structure-function relationship of GDE, the catalytic residues of yeast GDE were determined by site-directed mutagenesis. Asp-535, Glu-564, and Asp-670 on the N-terminal half and Asp-1086 and Asp-1147 on the C-terminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes. The five mutant enzymes, D535N, E564Q, D670N, D1086N, and D1147N were constructed. The mutant enzymes showed the same purification profiles as that of wild-type enzyme on ␤-CD-Sepharose-6B affinity chromatography. All the mutant enzymes possessed either transferase activity or glucosidase activity. Three mutants, D535N, E564Q, and D670N, lost transferase activity but retained glucosidase activity. In contrast, D1086N and D1147N lost glucosidase activity but retained transferase activity. Furthermore, the kinetic parameters of each mutant enzyme exhibiting either the glucosidase activity or transferase activity did not vary markedly from the activities exhibited by the wild-type enzyme. These results strongly indicate that the two activities of GDE, transferase and glucosidase, are independent and located at different sites on the polypeptide chain.

Molecular features of 23 patients with glycogen storage disease type III in Turkey: a novel mutation p.R1147G associated with isolated glucosidase deficiency, along with 9 AGL mutations

“…In addition, site-directed mutagenesis study in yeast glycogen debranching enzyme showed that amino acid substitutions in the C-terminal half lost glucosidase activity, but retained transferase activity. 23 Moreover, we expressed p.R1147G mutant and normal AGL proteins in COS cells transiently, and measured both glucosidase and transferase activities. 7 The p.R1147G exhibited negligible glucosidase activity, but retained 40% of transferase activity compared with wild AGL.…”

Hepatic and neuromuscular forms of glycogenosis type III: nine mutations inAGL

“…The mutated arginine p.Arg524His is located in exon 13, known to belong to the transferase domain, two aminoacid residues upstream from the aspartate which, in site-specific mutagenesis assays, was shown to alter the catalytic function of the domain itself (Nakayama et al, 2001).…”

Large-ring Cyclodextrins

“…In 2002, it was found that glycogen debranching enzyme (GDE, EC 2.4.1.25/EC 3.2.1.33) from Saccharomyces cerevisiae was also able to produce a LR-CD mixture (73). This enzyme has 4-α-glucanotransferase activity and amylo-1,6-glucosidase activity in the single polypeptide chain (74,75). GDE had some interesting effects on the LR-CD production, as follows: (1) The GDE could produce a LR-CD mixture containing CD11 to around CD40, from not only synthetic amyloses but also starches such as amylopectin and branched starch, and (2) there was no effect of the presence of D-glucose on the production of LR-CD mixture, because the GDE could not use D-glucose and maltose as acceptors of the coupling reaction.…”