Function of pyridoxal 5'-phosphate in glycogen phosphorylase: fluorine-19 NMR and kinetic studies of phosphorylase reconstituted with 6-fluoropyridoxal and 6-fluoropyridoxal phosphate

Abstract

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)

Evidence for Metabolism of Fluoromethyl 2,2-Difluoro-1-(trifluoromethyl)vinyl Ether (Compound A), a Sevoflurane Degradation Product, by Cysteine Conjugate β-Lyase

“…There was also similarity of the 2 J H-F coupling constant of the FCH 2 O group (54.6 and 54.9 Hz) and the 3 J H-F coupling constant of the CF 3 group (7.8 and 6.1 Hz) for the synthetic material and the metabolite. Signal broadening in the spectrum of the metabolite in urine compared to the synthetic material is consistent with previous 19 F NMR studies and has been ascribed to interaction with proteins (18). High urine protein concentrations are also known to occur in rats treated with FDVE (13).…”

Spectroscopic study of the Schiff bases of dodecylamine with pyridoxal 5′-phosphate and 5′-deoxypyridoxal. A model for the Schiff bases of pyridoxal 5′-phosphate in biological systems

“…Results shown here seem to be in disagreement with those coning fxom '9F-n.m.r. studies on phosphorylase b reconstituted with 6-fluoropyridoxal 5'-phosphate (6-FPLP) which have shown that the enzyme is active with the pyridine nitrogen in the neutral form and that the state of ionization of that nitrogen is unlikely to be affected by the binding of substrates Chang et al, 1986). The 6-FPLP-reconstituted phosphorylase b is very similar to the native phosphorylase but it was found to be far less active and similar to pyridoxal-reconstituted phosphorylase b, their VM,XJ values being 17.8, 66.7 and 15.4 ,umol/min per mg respectively (Chang et al, 1987).…”

“…The drastic change in the basicity of pyridine nitrogen in 6-FPLP (Korytnyk & Kravastava, 1973) will considerably alter the constant values in Scheme 2 in such a way that the binding of AMP and other substrates to 6-FPLP-reconstituted phosphorylase could not render the same coenzyme tautomer as in the native phosphorylase. The fact that the catalytic activity of the 6-FPLPreconstituted enzyme is not totally abolished has been taken as proof that the interaction between the pyridine nitrogen of PLP and the protein is not essential for catalytic activity and that the role of the ring nitrogen of PLP is structural rather than catalytic Chang et al, 1986Chang et al, , 1987). Nevertheless the difference in absorption spectrum observed on the binding of AMP and substrates to native phosphorylase cannot be explained if ionization changes on the coenzyme do not happen.…”

Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes

“…19F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'phosphate (AMP) complex shifted the 19F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate [Chang, Y. C., Scott, R. D., & Graves, D. J. (1986( ) Biochemistry 25, 1932( -1939. Analysis of the activation parameters, activation enthalpy and activation entropy, of the reaction of glycogen degradation catalyzed by phosphorylase containing pyridoxal phosphate, 6-FDPL, pyridoxal, or DPL showed that modifications of the coenzyme molecule affected only the activation entropy, not the activation enthalpy.…”