Study of the function of pyridoxal 5'-phosphate in glycogen phosphorylase

Abstract

Pyridoxal-reconstituted phosphorylase was used as a model system to study the possible functions of the 5'phosphoryl group of pyridoxal 5'-phosphate (PLP) in rabbit muscle glycogen phosphorylase. Kinetic studies were, conducted by using competitive inhibitors of phosphite, an activator, and a-D-glucopyranose 1-phosphate (glucose-l-P) to study the relationship between the PLP phosphate and the binding of glucose-l-P to phosphorylase. Fluorine-19 nuclear 19 magnetic resonance ( F NMR) spectroscopy of fluorophosphate bound to pyridoxal phosphorylase showed that its ionization state did not change during enzymatic catalysis. Evaluation of the apparent kinetic parameters for the activation of pyridoxal phosphorylase with different analogues having varied pK^g values demonstrated a dependency of on pK^2' Molybdate, capable of binding as chelates in a trigonal-bipyramidal configuration, was tested for its inhibitory property with pyridoxal phosphorylase. On the basis of the results in this study, several conclusions may be drawn: (1) The bound phosphite in pyridoxal phosphorylase and possibly, the 5'-phosphoryl group of PLP in native phosphorylase do not affect the glucose-l-P binding. (2) One likely function of the 5'-phosphoryl group of PLP in native phosphorylase is acting as an anchoring point to hold the PLP molecule and/or