Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites.

Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (Vderivative) and PA-0 (VPA-0), we demonstrated that the Vderivative/VPA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile.

Glycogen debranching pathway deduced from substrate specificity of glycogen debranching enzyme.

Glycogen debranching enzyme (GDE) is bifunctional in that it exhibits both 4-α-glucanotransferase and amylo-α-1,6-glucosidase activity at two distinct catalytic sites. GDE converts the phosphorylase-limit biantennary branch [G-G-G-G-(G-G-G-G↔)G-G- residue, where G = D-glucose, hyphens represent α-1,4-glycosidic bonds, and the double-headed arrow represents an α-1,6-glycosidic bond] into a linear maltooligosyl residue, which is then subjected to phosphorylase, and glycogen degradation continues. The prevailing hypothesis regarding the glycogen debranching pathway was that 4-α-glucanotransferase converts the phosphorylase-limit biantennary branch into the G-G-G-G-G-G-G-(G↔)G-G- residue and amylo-α-1,6-glucosidase cleaves the remaining α-1,6-linked G residue. In the present study, we analyzed the substrate specificities of 4-α-glucanotransferase and amylo-α-1,6-glucosidase using fluorogenic biantennary dextrins such as G-G-G-G-(G-G-G-G↔)G-G-GPA (F4/4/2; where GPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol), G-(G-G-G-G↔)G-G-GPA (F1/4/2), and G-G-G-G-G-G-G-(G↔)G-G-GPA (F7/1/2). Contrary to the prevailing hypothesis, the main branch of F4/4/2 was an important donor substrate component of 4-α-glucanotransferase and did not serve as an acceptor substrate. However, when G-G-G-G-G-GPA was added to the mixture, it successfully accepted a maltotriosyl (G3-) residue from F4/4/2. In addition, amylo-α-1,6-glucosidase exhibited strong activity towards G-G-G-G-(G↔)G-G-GPA but weak activity towards F7/1/2. Furthermore, the debranching activity of GDE towards phosphorylase-limit glycogen substantially increased when methyl α-maltooligosides with lengths equal to or greater than that of methyl α-maltopentaoside (G5-OCH3) were added to the enzyme reaction mixture. Based on these results, we propose the following macroscopic debranching pathway: Via 4-α-glucanotransferase, the G3- residue of the donor branch is transferred to a long (n ≥ 5) linear Gn- residue linked to a different branching G residue.