Synthesis and characterization of 6-fluoro 5'-deoxypyridoxal. Study of phosphorylase reconstituted with 6-fluoro 5'-deoxypyridoxal and 5'-pyridoxal

A new Vitamin B6 analogue, 6-fluoro 5'-deoxypyridoxal (6FDPL), was synthesized and characterized. Phosphorylase reconstituted with this analogue show 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical to those of phosphorylase reconstituted 6-fluoropyridoxal (6-FPAL), except the Km of phosphite toward the former enzyme was 9-times higher than that with the latter enzyme and the 6-FDPL-enzyme showed a lower V/sub max/ value. F-19 NMR studies showed that the binding of glucose-1-P to a 6-FDPL-enzyme-AMP complex shifted the F-19 signal 0.6 ppm toward upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed with glucose-1-P. Analysis of the activation parameters, of the glycogen breakdown reaction catalyzed by the native phosphorylase and phosphorylase containing 6-fluoropyridoxal 5'-phosphate (6-FPLD), 6-FPAL, 6-FDPL, pyridoxal or DPL showed that modifications of the coenzyme molecule only affected the activation entropy, not the activation enthalpy. These results suggest that the pyridine ring of the coenzyme may undergo a rotation during the course of catalysis; and the interaction between the coenzyme molecule with its neighboring amino acids are important to the efficiency of catalysis