Circular dichroism studies on glycogen phosphorylase from rabbit muscle. Interaction with the allosteric activator adenosine 5'-monophosphate

Abstract

Circular dichroism (CD) spectra of glycogen phosphorylase from rabbit muscle have been measured in the presence of various ligands, particularly in the near-ultraviolet wavelength region. Phosphorylases a and b gave similar positive CD spectra as each other, in the 250-310-nm region. The differences in CD between the a and b forms, as well as the CD changes induced by binding of substrate and other ligands except nucleotides to the enzyme, are all relatively small. Binding of AMP and other nucleotides to phosphorylases a and b, and NaBH4-reduced phosphorylase b, however, induces much larger CD spectral changes than the above. The difference CD curve obtained by subtracting the phosphorylase b curve from that of the enzyme- AMP complex is smooth, with a positive maximum at 266 nm and a negative at 289 nm. The results with various other nucleotides show that the induced Cotton effects are dependent on the base chromophore of the nucleotides. The rotational strength of the induced Cotton effect in phosphorylase b by AMP increases under various conditions, under which the affinity of the enzyme for AMP is enhanced, e.g., the addition of glucose 1-phosphate, inorganic phosphate, fluoride ion, divalent metal cations, and spermine, low temperatures, and conversion of the enzyme to the a form. On the contrary, these factors little affect the induced Cotton effects by IMP, GMP, and dAMP. Amylodextrin gave no effect on the extrinsic Cotton effect by binding of AMP plus Mn2+ to phosphorylase b, while it did retard the AMP-induced tetramerization of the enzyme. It is suggested that the interaction of nucleotides with phosphorylase involves stacking between the base ring of the bound nucleotides and an aromatic amino acid residue at the allosteric site of the enzyme, and that, in the high affinity form of the enzyme for AMP, particular bondings are newly formed between the enzyme and the nucleotide allowing the heterotropic cooperativity.