title: Purification and characterization of a glycogen phosphorylase analog missing the amino-terminal segment.
abstract: A glycogen phosphorylase analog missing only the amino-terminal 16 to 18 residues, which include the phosphorylation site, was produced by subtilisin Carlsberg cleavage of phosphorylase b in the presence of caffeine. The analog, named phosphorylase b's, was purified, and its enzymatic properties were compared with those of phosphorylase b. The KM's for glucose 1-phosphate are similar, but phosphorylase b's has a VM 43% higher than that of phosphorylase b. Also, phosphorylase b's is less sensitive to inhibition by glucose 6-phosphate and stimulation by sodium fluoride than is phosphorylase b. The subunit interactions in the two enzyme forms were also compared. The monomer-monomer interactions in phosphorylase b's are weaker than in phosphorylase b, as evidenced by a faster rate of resolution of the coenzyme, pyridoxal phosphate, from phosphorylase b's. The dimer-dimer interactions are also weaker in phosphorylase b's than in phosphorylase b, because phosphorylase b's does not form tetramers or crystals as readily as does phosphorylase b. Because removal of the amino-terminal segment changes the properties of the enzyme, this segment must be interacting with other parts of the protein. This statement conflicts with previous interpretation of X-ray crystallographic data that suggest that the amino-terminal region of phosphorylase b is freely mobile. Possible explanations for this contradiction are discussed.
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002110:Caffeine; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D010763:Phosphorylase b; D006005:Phosphorylases / Q000302:isolation & purification*; D013381:Subtilisins
chemical_list: D000596:Amino Acids; D002110:Caffeine; D010763:Phosphorylase b; D006005:Phosphorylases; D013381:Subtilisins