The substrate specificity of isoamylase and the preparation of apo-glycogenin

A new facet of the specificity of the glycogen-debranching enzyme, isoamylase, namely, the hydrolysis of a carbohydrate-amino acid linkage, is described. This bond joins the terminal, reducing-end D-glucose unit of glycogen to the hydroxyl group of tyrosine in glycogenin, the primer protein for glycogen biogenesis. The specificity was further defined by demonstrating that 4-nitrophenyl α-maltotrioside and higher homologs also act as substrates. The splitting of the glycogen-glycogenin bond by isoamylase indicates the α-anomeric configuration of the terminal D-glucose unit. It also provides a means of preparing apoglycogenin. Pullulanase, a somewhat similar starch- and glycogen-debranching enzyme, does not split these new isoamylase substrates, permitting the 4-nitrophenyl saccharides to be used in distinguishing between isoamylase and pullulanase.