Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG1, SG-1, SG-2, SG-3, SG-4, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG1 and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc m -AltNAc-Glc n -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG1 -SG-4 subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V derivative) with that toward PA-0 (V PA-0), we found that the value of V derivative/V PA-0 decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.
Keywords: Glycogen; Glycogen phosphorylase; Modified maltooligosaccharide; Pyridylamination; Substrate recognition.