How pyridoxal 5'-phosphate could function in glycogen phosphorylase catalysis.

A mechanism for the phosphorylase reaction is proposed which offers a plausible explanation for the essential role of pyridoxal 5'-phosphate in glycogen phosphorylases: in the forward direction, phosphorolysis of alpha-1,4-glycosidic bonds in oligo- or polysaccharides is started by protonation of the glycosidic oxygen by the substrate orthophosphate followed by stabilization of the incipient oxocarbonium ion and subsequent covalent binding to form alpha-glucose 1-phosphate. In the reverse direction, protonation of the phosphate of glucose 1-phosphate destabilizes the glycosidic bond and promotes formation of a glucosyl oxocarbonium ion-phosphate anion pair. In the subsequent step the phosphate anion facilitates the nucleophilic attack of a terminal glucosyl residue on the carbonium ion bringing about alpha-1,4-glycosidic bond formation and primer elongation. Both in the forward and reverse reactions, the phosphate of the cofactor pyridoxal 5'-phosphate acts as a general acid (PL-OPO3H- or PL-OPO3(2-) and protonates the substrate phosphate functioning as proton shuttle. Thus in glycogen phosphorylases, phosphates which directly interact with each other have replaced a pair of amino acid carboxyl groups functioning in catalysis of carbohydrases.     

Structural changes accompanying the removal of pyridoxal 5'-phosphate from phosphorylase b.

The changes in physical properties accompanying the removal of pyridoxal 5'-phosphate from glycogen phosphorylase b have been examined. The apoenzyme retains a high degree of structural rigidity, as determined from the time decay of anisotropy. The bulk of the secondary structure remains intact, although a significant change in circular dichroism indicates some degree of alteration. The mobility of a sulfhydryl-linked spin label increases. The restoration of pyridoxal 5'-phosphate reverses this effect, with indication of interaction between subunits. One or more new binding sites for 1-anilinonaphthalene-8-sulfonate appear for the apoenzyme. The kinetics of the recombination of pyridoxal 5'-phosphate with the apoenzyme, as monitored by difference spectra, indicate a high activation energy for the process. The apoenzyme is a reversibly associating system at 20-30 degrees C, pH 7.0.     

Influence of pH on the removal of pyridoxal 5'-phosphate from phosphorylase b

A method to break the pyridoxal 5'-phosphate (PLP)-phosphorylase b bond using hydroxylamine and slightly acid pH is put forward and described in the present paper. This method does not involve drastic conditions or deforming reagents. The influence of pH and protein concentration on the removal of PLP from phosphorylase has also been studied, resulting in an order of -0.3 with respect to the enzyme, a value that implies a complex reaction. An additional conclusion is that an increase in the protein concentration entails better protection of the enzyme from attack by hydroxylamine.     

Function of the phosphate group of pyridoxal 5'-phosphate in the glycogen phosphorylase reaction

To understand the catalytic mechanism of glycogen phosphorylase (EC 2.4.1.1), pyridoxal(5')phospho(1)-beta-D-glucose was synthesized and examined as a hypothetical intermediate in the catalysis. Pyridoxal phosphoglucose bound stoichiometrically to the cofactor site of rabbit muscle phosphorylase b in a similar mode of binding to the natural cofactor, pyridoxal 5'-phosphate. The rate of binding of pyridoxal phosphoglucose was only 1/100 compared with that of pyridoxal phosphate. The enzyme reconstituted with pyridoxal phosphoglucose showed no enzymatic activity at all even after prolonged incubation of the enzyme with substrates and activator. The present data would contradict participation of the phosphate group of pyridoxal phosphate in a covalent glucosyl-enzyme intermediate even if the covalent intermediate was formed during the catalysis.     

Dissociative mechanism of thermal denaturation of rabbit skeletal muscle glycogen phosphorylase b

The thermal stability of rabbit skeletal muscle glycogen phosphorylase b was characterized using enzymological inactivation studies, differential scanning calorimetry, and analytical ultracentrifugation. The results suggest that denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monomers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibitor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that the cofactor of glycogen phosphorylase-pyridoxal 5'-phosphate-stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheated at 53 degrees C occurs after cooling of the enzyme solution to 30 degrees C. The fact that the rate of reactivation decreases with dilution of the enzyme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phosphorylase b reactivation.     

Regulation of the activity of rabbit skeletal muscle phosphorylase kinase by glycogen

The effects of glycogen on the non-activated and activated forms of phosphorylase kinase were studied. It was found that in the presence of glycogen the activity of non-activated kinase at pH 6.8 and 8.2 and that of the activated (in the course of phosphorylation) form are enhanced. The degree of activation depends on glycogen concentration. At saturating concentrations, this enzyme activity increases 2-3-fold; the enzyme affinity for the protein substrate, phosphorylase b, also shows an increase. The polysaccharide has no effect on the activity of phosphorylase kinase stimulated by limited proteolysis. In the presence of glycogen, the rate of autocatalytic phosphorylation of the enzyme is increased. Glycogen stabilizes the enzyme activity upon dilution. The experimental results suggest that the polysaccharide directly affects the phosphorylase kinase molecule. The maximal binding was shown to occur at the enzyme/polysaccharide ratio of 1:10 (w/w) in the presence of Ca2+ and Mg2+.     

The pyridoxal 5' -phosphate site in rabbit skeletal muscle glycogen phosphorylase b: an ultraviolet and 1H and 31P nuclear magnetic resonance spectroscopic study.

1 H NMR spectra of the 3-0-methylpyridoxal 5'-phosphate-n-butylamine reaction product indicated that this analogue forms a Schiff base in aprotic solvent. The uv spectral properties of 3-0-methylpyridoxal-5'-phosphate phosphorylase b correspond to those of the n-butylamine Schiff base derivative in dimethyl sulfoxide. On the basis of that and auxiliary uv and 1H NMR spectra of pyridoxal and pyridoxal 5'-phosphate and the corresponding Schiff base derivatives we have verified that pyridoxal 5' -phosphate is also bound as a Schiff base to phosphorylase and not as an aldamine. Since 3-0-methylpyridoxal-5'-phosphate phosphorylase is active, a proton shuttle between the 3-hydroxyl group and the pyridine nitrogen is excluded. This directs attention to the 5' -phosphate group of the cofactor as a candidate for a catalytic function. 31P NMR spectra of pyridoxal 5' -phosphate in phosphorylase b indicated that deprotonation of the 5' -phosphate group was unresponsive to external pH. Interaction of phosphorylase b with adenosine 5' -monophosphate, the allosteric effector required activity, and arsenate, which substitutes for phosphate as substrate, triggered a conformational change which resulted in deprotonation of the 5' -phosphate group of pyridoxal 5' at pH 7.6. It now behaved like in the pyridoxal-phosphate-epsilon-aminocaproate Schiff base in aqueous buffer, where the diionized form is dominant at this pH. Differences of line widths of the adenosine 5' -monophosphate signal point to different life times of the allosteric effector- enzyme complexes in the presence and absence of substrate (arsenate).     

Relationships between dephosphorylation and D to I conversion of rabbit skeletal muscle glycogen synthase.

The relationship between dephosphorylation and D to I conversion of skeletal muscle glycogen synthase by synthase phosphatase was investigated using synthase preparations containing 1 to 3 mol of 32P/mol of subunit (90,000 g). Dephosphorylation was analyzed in terms of 32P release from the trypsin-sensitive and trypsin-insensitive phosphorylation regions of synthase. With synthase containing 1 to 2 mol of 32P/90,000 g, dephosphorylation of the trypsin-insensitive region correlated closely with D to I conversion and was more rapid than dephosphorylation of the trypsin-sensitive region. Synthase containing 3 mol of 32P/90,000 g was a relatively poor substrate for the phosphatase since dephosphorylation of both regions, as well as D to I conversion, was slow. With this species of synthase, glucose-6-P (0.1 mM) increased the rates of D to I conversion and dephosphorylation of trypsin-insensitive region. It is concluded that dephosphorylation of the trypsin-insensitive region is responsible for the conversion of synthase D to I.