Structural Investigations on Glycogen Debraching Enzyme

Structural investigations on glycogen metabolizing enzymes are restricted to glycogen phosphorilase, despite the physiologic importance of other enzymes witnessed by association of Glycogen Storage Diseases (GSD) with their deficiency. Type III GSD (Cori’s disease is due to the absence of Glycogen Debranching Enzyme (GDE). These reason prompted a study of structural features of GDE, by combining limited proteolysis, spectroscopic and calorimetric approaches. Cleavage by V8 proteinase releases small fragments with only slight decline in molecular size of GDE protein. Extensive cleavage was obtained with chimotrypsin and trypsin, with similar release of 3 main peptides, and preservation of catalytic activity. We have determined the secondary structure of the intact and of the cleaved protein and of the peptides isolated in buffers of high ionic strength. By differential scanning calorimetry we have detected 3 melting transitions very close to each other (Tm between 54 and 60 C°), which are partially reproduced during thermal unfolding of isolated peptides. Our data support the hypothesis of a complex structure of GDE with 3 main compact regions corresponding to the N- and C-terminal separated by an intermediate region, each containing about 500 AA. The N-terminal is relevant for the transferase activity, while the glucosidase activity is located in the C-terminal region. Notably, interruption of amminoacids sequence does not affect the overall conformation of the protein.