The role of pyridoxal 5'-phosphate in plant phosphorylase.
The removal of pyridoxal 5'-phosphate from potato phosphorylase [EC 2.4.1.1] was achieved by incubation in an acidic ammonium sulfate solution containing hydroxylamine. Potato apophosphorylase is catalytically inactive, but reactivated by incubation with pyridoxal phosphate. Upon titration, the degree of recovery of activity agreed well with the degree of incorporation of pyridoxal phosphate. Therefore, it can be concluded that pyridoxal phosphate in the plant enzyme is the cofactor required for the enzyme activity, as it has been shown to be in the animal enzyme. The reconstitution of the apoenzyme using six pyridoxal phosphate analogues modified at the 5' position indicates that the structural properties of the cofactor binding site is similar in potato and rabbit muscle phosphorylases. This suggests that the plant enzyme has also a substrate binding site neighboring with the 5'-phosphate moiety of the bound cofactor similar to that shown to exist in the animal enzyme. On the other hand, the analysis of the far-ultraviolet circular dichroism spectra of these two enzymes shows that the secondary structures are rather different from each other; the potato enzyme is estimated to have 16% alpha-helix and 37% beta-structure, and the muscle enzyme b 41% alpha-helix and 21% beta-structure. This indicates that the cofactor binding locus must have been more strictly conserved than other regions.
D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D002942:Circular Dichroism; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D006005:Phosphorylases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011817:Rabbits; D013045:Species Specificity
D001051:Apoenzymes; D006898:Hydroxylamines; D011732:Pyridoxal Phosphate; D006005:Phosphorylases; D000645:Ammonium Sulfate