IONIZATION OF PYRIDOXAL 5'-PHOSPHATE AND THE INTERACTIONS OF AMP-S AND THIOPHOSPHOSERYL RESIDUES IN NATIVE AND SUCCINYLATED RABBIT MUSCLE GLYCOGEN PHOSPHORYLASE-B AND PHOSPHORYLASE-A AS INFERRED FROM P-31 NMR-SPECTRA

Rabbit skeletal muscle phosphorylase b or a dissociates on treatment with succinic anhydride, yielding a mixture of about 40% monomer and 60% dimer with a residual activity of about 15%. The phosphate signal of pyridoxal phosphate in the native, inactive form of phosphorylase b in the absence of ligands was assigned to a shielded protonated form. The same resonance line with the identical chemical shift was observed in a monomer-dimer mixture of succinylated phosphorylase b, indicating that dissociation had no effect on the ionization of the phosphate group of pyridoxal phosphate. This is not compatible with a location of the cofactor at or near the subunit interface. Glucose, an inhibitory substrate analogue, binds at the α-glucose 1-phosphate site. Experiments with pyridoxal 5‘-deoxymethylenephosphonate reconstituted phosphorylase a have shown that the 5‘ side chain of the cofactor analogue reports the binding of glucose. These findings were interpreted in support of a pyridoxal phosphate location near the catalytic site. Binding of the allosteric effector adenosine 5‘-O-thiomonophosphate (AMP-S) to succinylated phosphorylase b caused the protonated form of the cofactor to decline in favor of the dianionic form, the amount of which correlates with the dimer concentration. In native phosphorylase a, the phosphate of pyridoxal phosphate is in the dianionic form, whereas the main phosphate signal in succinylated phosphorylase a represents the protonated form. Addition of AMP-S to the latter leads to an increase in the dianionic form, but a complete conversion was not achieved. At pH 6.5, the optimum for activity, the dianionic thiophosphoseryl residue of native phosphorylase a forms a stable salt bridge. At more alkaline pH (7.7), attachment of the thiophosphoseryl residue is partly abolished, but this residue can be reattached on adding AMP-S, indicating that the phosphates of the nucleotide and of the phosphoseryl residue bind to different but cooperative sites. The succinylated AMP-S phosphorylase a complex has a much shorter lifetime even at a more acid pH (7.35) than the corresponding complex with native phosphorylase a at pH 7.67, leaving most of the thiophosphoseryl side chains mobile. This might be related to the much greater dependence of succinylated phosphorylase a for adenosine 5‘-monophosphate to express activity. © 1979, American Chemical Society. All rights reserved.