Paradoxical Inhibition of Phosphorylase by Pyridoxal Phosphate

The reaction of phosphorylase b with pyridoxal-P results in partial inactivation of the enzyme and a change in enzymic characteristics. The reaction of enzyme with pyridoxal-P is reversible; it can be changed to an irreversible reaction by reduction of the pyridoxal-P-phosphorylase b complex with sodium borohydride. Subsequent analysis of the pyridoxalP-phosphorylase b derivative shows that the major changes in enzymic characteristics are primarily due to modification of one group per monomer at a specific site on the enzyme. The unreduced pyridoxal-P-phosphorylase b complex shows a difference spectrum with a maximum at 432 nm, which is characteristic for the Schiff base formation that follows phosphorylase b-pyridoxal-P interaction. After reduction with sodium borohydride the absorption spectrum shows a maximum at 325 nm, which is typical for e-phosphopyridoxallysine. Fluorescence spectrometry was used to measure the initial rate of reaction of pyridoxal-P with phosphorylase b to form the Schiff base. This rate as a function of pyridoxal-P concentration was a rectangular hyperbola, indicating that the reagent was initially absorbed to a site on the enzyme with an apparent dissociation constant of 8 x 10-4 m. Schiff base formation followed. AMP plus glucose-1-P and ATP show protective effects against the modification by pyridoxal-P, whereas AMP, glucose-1-P, Pi, and glycogen showed little effect. Pyridoxal and other analogs tested for their effect on phosphorylase b have not been found as effective as pyridoxal-P. Imidazole citrate, which causes dissociation of phosphorylase b, does not affect the rate of reaction of pyridoxal-P and phosphorylase b. The pyridoxal-P-phosphorylase b derivative, which is still active at high substrate concentration, can be treated to remove the original native pyridoxal-P coenzyme without affecting the extra covalently attached pyridoxal-P. This apo form of the pyridoxal-P-phosphorylase b derivative is inactive. Phosphorylase a may also incorporate 1 to 2 molecules of pyridoxal-P per monomer, resulting in a partially inactivated enzyme with properties somewhat different from the pyridoxal-P-phosphorylase b derivative. The chemical binding site for pyridoxal-P in the latter derivative has been identified as the e-amino group of a lysine residue.