Function of pyridoxal 5'-phosphate in glycogen phosphorylase: 19F NMR and kinetic studies of phosphorylase reconstituted with 6-fluoropyridoxal and 6-fluoropyridoxal phosphate.
19F NMR spectroscopic properties of glycogen phosphorylase reconstituted with 6-fluoropyridoxal (6-FPAL) and 6-fluoropyridoxal phosphate (6-FPLP) were investigated. Analysis of the contribution of chemical shift anisotropy to the line width of the 6-FPLP-enzyme signal shows that the coenzyme molecule is tightly bound to the protein. The chemical shift of the fluorine nucleus in the free 6-FPLP protein is pH independent from pH 6 to pH 9.1. When the 6-FPLP-enzyme forms complexes with AMP, AMP plus glucose-1-P, and AMP plus inorganic phosphate, signals at -11.0, -13.1, and -10.4 ppm are observed, respectively. These different chemical shifts indicate that the protein in each complex has a distinct conformation. The exchange rate between the 6-FPLP-protein-AMP complex and the same complex with bound glucose-1-P is estimated to be 3300 +/- 700 s-1, and that between the 6-FPLP-protein-AMP complex and with bound inorganic phosphate is 500 +/- 100 s-1. The former exchange rate is 13 times faster than that of the same process for the 6-FPAL-enzyme. Analysis of the effects of temperature on the 19F line shape of the 6-FPLP enzyme in the presence of ligands shows that the exchange rates between different complexes drop significantly between 20 and 10 degrees C. Within this temperature range, Arrhenius plots of the enzymatic activities of the native and 6-FPLP-enzymes at varied temperatures also show a pronounced curvature.(ABSTRACT TRUNCATED AT 250 WORDS)
Y C
YC
Chang
R D
RD
Scott
D J
DJ
Graves
D001665:Binding Sites; D005461:Fluorine; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D006005:Phosphorylases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011730:Pyridoxal / Q000031:analogs & derivatives* / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D013816:Thermodynamics
D005461:Fluorine; D011730:Pyridoxal; C044385:6-fluoropyridoxal; D011732:Pyridoxal Phosphate; C044386:6-fluoropyridoxal phosphate; D006005:Phosphorylases