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Mechanistic studies of rabbit muscle glycogen phosphorylase Stirtan, William G.

Abstract

Glycogen phosphorylase catalyzes the reversible phosphorolysis of glycogen. The enzyme contains a molecule of pyridoxal phosphate (PLP), of which the phosphate moiety is essential for catalysis. To investigate the role of the cofactor phosphate moiety two phosphonate analogues of PLP, 5'-deoxypyridoxal-5'-methylenephosphonic acid (9) and5'-deoxypyridoxal-5'-difluoromethylene phosphonic acid (13), were prepared and reconstituted into apoglycogen phosphorylase b. Both such reconstituted enzymes had activities of approximately 25% - 30% of the native enzyme, and moreover, despite the considerable difference in cofactor pKa, the pH-dependences of Vmax, Km and Vmax/Kmfor the two enzyme systems were quite similar. These results suggest that PLP does not function as an essential acid catalyst in glycogen phosphorylase; rather they suggest that the cofactor phosphate remains dianionic throughout catalysis. Evidence concerning the protonation state of the PLP phosphate moiety within the R- and T-state enzyme conformations was obtained from solid-state magic angle spinning 31P NMR experiments of the native enzyme. The cofactor phosphate moiety, in both allosteric forms of the enzyme, exhibited axially symmetric shielding parameters. These results suggest that the PLP phosphate moiety exists as a dianion in both enzyme conformations, and therefore, that a change in protonation state does not accompany the allosteric transition. The compound, 1-nitro-D-glucal, was found to irreversibly inactivate both the R-and T-state forms of glycogen phosphorylase, behaving as an active site-directed affinity label. The pH-dependence of the inactivation was consistent with 1-nitro-D-glucalundergoing a conjugate addition reaction at the C-2 position with an active site nucleophile, possibly Tyr-573. X-ray structural studies of the inactivated T-state enzyme identified Tyr-573 as a likely candidate for the inactivation, and further, identified a surface amino acid (His-73) covalently bonded to the C-2 position of a second molecule of the affinity label, the product of a conjugate addition reaction. The cofactor-substrate analogues, pyridoxa1-5'-pyrophospho-1-a-D-glucose(PLPP-Glu) (21) and pyridoxa1-5'-pyrophospho-1-(2-deoxy-2-fluoro)-a-D-glucose(PLPP-2FG1u) (23), were prepared and reconstituted into apoglycogen phosphorylase b.Results from kinetic, 19F NMR and cofactor abstraction studies demonstrated that PLPP-2FG1u, unlike PLPP-Glu, is completely resistant to decomposition at the enzyme active site, and furthermore, does not transfer its glucosyl moiety to glycogen. Glycogen phosphorylase reconstituted with PLPP-2FG1u has allowed crystallization and initial structural analysis of the activated enzyme / substrate complex. The solution binding interaction between glycogen and phosphorylase has been probed by 19F NMR using a glycogen analogue in which all the non-reducing terminal glucosyl residues have been replaced by 4-deoxy-4-fluoro-glucosyl moieties (4-F-glycogen). Results from titrating the fluorinated glycogen analogue with phosphorylase suggest that 60 phosphorylase dimers bind per glycogen particle. 4-F-glycogen has also been investigated as an "incompetent" substrate analogue in an attempt to provide evidence for a glucosyl-enzyme intermediate in phosphorylase. Results from kinetic and radiolabelling studies demonstrated that the fluorinated glycogen analogue used was not completely inert to glucosyl transfer, but rather, that it possessed approximately 1% of the activity observed with normal glycogen, most likely due to incomplete derivatization of the glycogen.

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