Does pyridoxal 5'-phosphate function in glycogen phosphorylase as an electrophilic or a general acid catalyst?

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Abstract
.alpha.-D-Glucose 1-diphosphate interacts with pyridoxal-reconstituted rabbit muscle phosphorylase b activated by AMP (AMP-S). Under these conditions, the glucose moiety of .alpha.-D-[14C]glucose 1-diphosphate is transferred to limit dextrin forming .alpha.(1 .fwdarw. 4) glycosidic bonds and simultaneously releasing pyrophosphate as shown by 31P NMR spectroscopy. Thus, specific structural requirements invoked to explain the reactions of pyridoxal(5'')diphospho(1)-.alpha.-D-glucose need not to be assumed in the case of the reactions of .alpha.-D-glucose 1-diphosphate. Dianions isomorphous to phosphate activate pyridoxal phosphorylase regardless of their pK values while the same anions, when bound covalently to pyridoxal, are inactive. Thus, anions bound noncovalently to pyridoxal phosphorylase act differently than anions linked covalently to pyridoxal, such as the 5''-phosphate group of pyridoxal 5''-phosphate, which is postulated to be part of a proton donor-acceptor pathway. The reaction of 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) with phosphorylase yields, in the presence of orthophosphate as a glycosyl acceptor, 1-deoxy-D-gluco-heptulose 2-phosphate (heptulose-2-P). This sugar phosphate is unreactive but a potent competitive inhibitor for rabbit muscle phosphorylase b and potato phosphorylase with respect to .alpha.-D-glucose 1-phosphate: Ki = 14 .times. 10-6 M and 1.9 .times. 10-6 M, respectively. Heptulose-2-P is ideally suited for 31P NMP experiments with phosphorylase because its phosphate resonance and that of the 5''-phosphate or pyridoxal 5''-phosphate do not overlap, and in contrast to .alpha.-D-glucose-pyranose cyclic 1,2-phosphate, another powerful inhibitor of glycogen phosphorylases, heptulose-2-P is protonatable in the pH range of the enzymatic reaction. Use was made of this property of heptulose-2-P in 31P NMR investigations, the results of which indicated that the dianionic 5''-phosphate group of the natural cofactor becomes partially protonated on binding of heptulose-2-P to potato phosphorylase, whereas the monoprotonated 5''-phosphonate group of the partially active 5''-deoxypyridoxal-5''-(methylenephosphonate) muscle phosphorylase b derivative was shown to share a proton with the phosphate moiety of heptulose-2-P. While the experiments support a role for the cofactor phosphate in glucogen phosphorylases in a general acid-base catalysis, they do not uphold a function of the phosphorus of the 5''-phosphate group of the cofactor as an electrophilic catalyst.