Direct phosphate-phosphate interaction between coenzyme and substrate in the catalytic reaction of glycogen phosphorylase

Glycogen phosphorylase contains firmly bound pyridoxal 5′-phosphate (PLP), and catalyzes the reversible transfer of a glucosyl moiety between glucose-1-phosphate (G-1-P) and α-1,4-glucan. X-ray crystallographic studies revealed that PLP is located in a pocket where the phosphate group of PLP is pointed toward the G-1-P binding site. We have synthesized pyridoxal(5′)diphospho(1)-α-d-glucose, as a model compound for the phosphate-phosphate interaction between PLP and G-1-P, and reconstituted the enzyme with this compound. The resulting enzyme is catalytically inactive in itself, but, in the presence of glucan, the glycosyl moiety of this compound is transferred to the glucan forming a new α-1,4-glucosidic linkage along with the production of pyridoxal 5′-diphosphate. This glucosyltransfer is similar to the normal catalytic reaction in various aspects, although the rate is smaller in the order of three. AMP accelerates the transfer about 24 times compared with the reaction in its absence. We have more recently used pyridoxal(5′)triphospho(1)-α-D-glucose to reconstitute the enzyme. In the presence of glucan, the compound bound to enzyme is gradually degraded to pyridoxal 5′-triphosphate. This reaction is essentially dependent on AMP, and proceeds several times more slowly than the glucosyltransfer from the diphospho compound. These results provide evidence for the direct phosphate-phosphate interaction between the coenzyme and the substrate in the normal enzyme reaction, and seem to reflect a rather wide allowance in regard to this interaction.     

Effect of 1,2-dimethoxyethane on potato phosphorylase for polysaccharide specificity

Potato phosphorylase normally utilizes starch as a substrate but will not use glycogen effectively. In the presence of 20% 1,2-dimethoxyethane, this specificity is lost because of marked activation with glycogen and some inhibition with starch. The effect of dimethoxyethane on β-amylase was the same for both starch and glycogen. These was no contaminating enzyme that would make glycogen more “starch-like.” The 20-fold decrease in K_m for glycogen, a change in pH profile, and elimination of inhibition by cyclodextrin suggest that dimethoxyethane causes a change in phosphorylase structure.     

Effect of glucose-6-P on the catalytic and structural properties of glycogen phosphorylase a

A monoclonal antibody to porcine beta-lipotropin has been produced which binds to the N-terminal (gamma-lipotropin) portion of the molecule. The antibody can be used to detect beta-lipotropin as well as other beta-endorphin precursors (predominantly a Mr 38 000 polypeptide) using radiobinding assay or the immunoblotting technique. Purification of the peptides can be readily achieved by affinity chromatography using the monoclonal antibody covalently bound to Sepharose 4B. As the antibody recognises the N-terminal part of beta-lipotropin, it can be used to detect and purify beta-lipotropin and other beta-endorphin precursors in the presence of beta-endorphin.     

Purification and properties of glycogen phosphorylase from Streptococcus salivarius

Glucose-grown cells of Streptococcus salivarius have been shown to contain a polyglucose phosphorylase which had maximum activity in the stationary phase of growth. Despite the fact that activity in crude cell-free extracts was two- to threefold greater in the presence of corn dextrin than with oyster glycogen, subsequent purification (200-fold) of the enzyme from the soluble fraction of the organism by protamine sulfate treatment, ammonium sulfate fractionation (30–50%), ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 demonstrated that this dextrin/glycogen activity was associated with a single enzyme. Since glucose-grown cells of S. salivarius are known to synthesize a typical glycogen polymer, the enzyme was named: glycogen phosphorylase. The purified enzyme preparation was devoid of phosphoglucomutase and ADP-glucose pyrophosphorylase, but contained a small amount of ADP-glucose: α-1,4 glucan transferase activity. The enzyme was stable at ?10 °C in the presence of 0.2 m NaF, while the pH optimum for the enzyme was 6.0 both with glycogen and with dextrin. With the purified enzyme, corn dextrin was the best primer, both in the direction of synthesis and in the direction of phosphorolysis, being 1.8–1.9 times more effective than purified S. salivarius glycogen. When the enzyme was assayed in the direction of glycogen synthesis, a K_m value of 3.4 mm was obtained for glucose-1-P, while the values for S. salivarius glycogen, oyster glycogen and corn dextrin were 25, 42, and 40 mg/ml, respectively. In the direction of phosphorolysis, K_m values were 20 mm for P_i obtained with oyster glycogen, 25 mm for P_i with corn dextrin, and 20 mg/ml and 26 mg/ml for oyster glycogen and corn dextrin, respectively. Present data suggests no involvement of -SH groups in enzyme catalysis, while the enzyme was inhibited by divalent ions with the severest inhibition being observed with Ca²⁺, Zn²⁺ and Fe²⁺. The two ion chelators, EDTA and EGTA, had no effect on enzyme activity.     

Inhibition of muscle glycogen phosphorylase b by vitamin B2 and its coenzyme forms

Kinetic studies have demonstrated that vitamin B2 and its coenzyme forms FMN and FAD are potent inhibitors of glycogen phosphorylase b from rabbit skeletal muscle. The inhibition of the enzyme by flavins has a co-operative character (Hill coefficients exceed unity). Glycogen phosphorylase b bound to FMN or FAD does not reveal catalytic activity, whereas the enzyme bound to riboflavin retains about 16% of the initial catalytic activity.     

Binding of vitamin B2 and its coenzyme forms by muscle glycogen phosphorylase b

Binding of vitamin B2 and its coenzyme forms by glycogen phosphorylase b was studied by sedimentation velocity and sedimentation equilibrium methods. Microscopic dissociation constants for complexes of the enzyme with riboflavin, FMN and FAD were found to be 12.5, 6.8 and 18.1 microM, respectively (0.1 M KCl, pH 6.8, 20 degrees C). We revealed also that glucose 1-phosphate, glycogen and AMP decreased the affinity of the enzyme for FMN.     

Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.     

Some regulatory properties of glycogen phosphorylase from Streptococcus salivarius

Purified (200-fold) glycogen phosphorylase (EC 2.4.1.1) of Streptococcus salivarius was activated by AMP and NaF when assayed both in the direction of synthesis and in the direction of phosphorolysis. Activation by NaF + AMP was greater than the sum of their individual effects. In the direction of synthesis, the K_m for AMP was 0.25 mm and was decreased to 0.125 mm in the presence of NaF. The K_m for NaF was 0.49 m and was decreased to 0.40 m in the presence of AMP. Glycogen phosphorolysis was similarly affected by AMP and NaF, except that above a concentration of 2 mm AMP was inhibitory. The effects of AMP and NaF were reversible since preincubation with these compounds, followed by dialysis, restored activity almost to the control values although some inhibition of enzyme activity was noted with the samples preincubated with NaF. The presence of both NaF and AMP had no effect on the K_m values for glucose-1-P and glycogen in the direction of synthesis, but increased the V of the enzyme.When assayed in the absence of AMP and NaF in the direction of synthesis, the enzyme was slightly inhibited by glucose and glucose-6-P, and activated by P-enolpyruvate and ADP-glucose. In the presence of AMP and NaF, the enzyme was inhibited by glucose, glucose-6-P and ADP-glucose, but was activated by P-enolpyruvate. Fructose-1,6-P₂ had no effect on the enzyme. The enzyme was further activated in the absence of AMP and NaF by adenosine, ATP, GMP, cyclic AMP and ADP, and was slightly inhibited by GTP and GDP. In the presence of AMP and NaF, however, these compounds, with the exception of adenosine, either did not show any effect or were slightly inhibitory. Adenosine was slightly stimulatory with NaF + AMP, but not with AMP alone. In the direction of phosphorolysis, the enzyme was inhibited by glucose and ADP-glucose, and activated by P-enolpyruvate, fructose-1,6-P₂ and ATP, both in the presence and absence of AMP + NaF.     

Interaction between glycogen and glycogen phosphorylase of Tetrahymena

The glycogen phosphorylase of Tetrahymena pyriformis complexes with glycogen as judged by its elution pattern from columns of Sepharose 6B. Complex formation does not occur with starch, amylose, or amylopectin, and neither do these polyglucans serve as primers for the enzyme. To study the association between the phosphorylase and glycogen particles in situ, Tetrahymena were grown under differing physiological conditions, phosphorylase was isolated and chromatographed on a Sepharose 6B column. Phosphorylase activity isolated from cells grown in the absence of glucose was only partially associated with glycogen, while in cells exposed to glucose for 30 min or more all the phosphorylase activity was associated with glycogen. The effects of culture age and anaerobiosis on the relative amounts of free and glycogen-bound enzyme in the cells were also studied. It was concluded from the in vivo experiments that there was no simple relation between the fraction of enzyme bound to glycogen and between cell glycogen content.     

Effects of lithium ions on glycogen synthase and phosphorylase in rat hepatocytes

Incubation of hepatocytes from fasted rats with LiCl provoked a concentration- and time-dependent activation of glycogen synthase. This effect was observed in the absence of glucose in the incubation medium. No changes in the intracellular concentrations of ATP or glucose-6-phosphate were detected. Lithium was also able to activate glycogen synthase in the absence of extracellular calcium. If hepatocytes were incubated with lithium and insulin, an additive effect of both agents on glycogen synthase activity was observed. LiCl was also effective in activating the enzyme in hepatocytes obtained from fed rats. When hepatocytes were incubated with [33P]phosphate and then treated with LiCl, a decrease in the amount of [32P]phosphate incorporated in the enzyme was observed. This dephosphorylation affected two CNBr fragments of the enzyme (CB-2 and CB-1), suggesting that several phosphorylation sites were involved. Lithium was also able to activate glycogen phosphorylase from both fasted and fed rats. Phosphorylase activation was concentration- and time-dependent, either in the presence or absence of calcium in the incubation medium. These findings demonstrate that although lithium appears to mimic the effects of insulin on glycogen synthase activity, its mechanism of action must be different from that of the hormone.     

Use of 6-fluoroderivatives of pyridoxal and pyridoxal phosphate in the study of the coenzyme function in glycogen phosphorylase

6-Fluoropyridoxal phosphate (6-FPLP) has been synthesized. Its properties were studied, and it was used, along with 6-fluoropyridoxal (6-FPAL), to reconstitute apophosphorylase b. Kinetic studies of the resulting enzymes showed that phosphorylases reconstituted with 6-FPLP and 6-FPAL have characteristics similar to those of native and pyridoxal enzymes, respectively, except that the former two enzymes have lower Vmax values. 19F NMR and UV spectra of 6-FPLP phosphorylase showed that the coenzyme forms a neutral enolimine Schiff base. Because the UV and fluorescence spectra of 6-FPLP phosphorylase are comparable to those obtained with native phosphorylase, it further confirms the postulate that pyridoxal phosphate forms a neutral enolimine Schiff base in phosphorylase. The results suggest that the 3-OH group is protonated and the pyridine nitrogen unprotonated in both 6-FPLP phosphorylase and native enzyme. 19F NMR study of 6-FPLP- and 6-FPAL-reconstituted phosphorylases in the inactive and active states indicates that the protein structure near the coenzyme binding site undergoes certain changes when these enzymes are activated by the substrates and AMP. The comparison of the properties of 6-FPLP-reconstituted and native phosphorylases implies that the ring nitrogen of the coenzyme PLP in phosphorylase may interact with the protein during catalysis, and this interaction is important for efficient catalysis by phosphorylase.