Phosphonate and alpha-fluorophosphonate analogue probes of the ionization state of pyridoxal 5'-phosphate (PLP) in glycogen phosphorylase.

To investigate the role of the essential cofactor pyridoxal phosphate in rabbit muscle glycogen phosphorylase catalysis, two phosphonate analogues of pyridoxal phosphate, 5'-deoxypyridoxal 5'-methylenephosphonic acid and 5'-deoxypyridoxal 5'-difluoromethylenephosphonic acid, have been prepared and reconstituted into apophosphorylase b. UV/Vis spectroscopic and 31P and 19F NMR studies confirmed the successful reconstitution and revealed significant changes in phosphate environment upon nucleotide activation. Both such reconstituted enzymes had activities of approximately 25%-30% of that observed in the native enzyme, while K(m) values were similar to those of the native enzyme. Very similar dependences upon pH of Vmax, K(m), and Vmax/K(m) were found for the two reconstituted enzyme derivatives and the native enzyme despite the considerable difference in phosphonic acid pKa values. These results suggest that pyridoxal phosphate does not function as an essential acid/base catalyst in glycogen phosphorylase; rather they suggest that the cofactor phosphate moiety remain dianionic throughout catalysis and functions as an essential dianion. Mechanistic implications of these findings are discussed.