Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.     

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.     

Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein

Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5'-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (> or = 50 degrees C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate approximately 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the alpha-helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5'-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to approximately 50 degrees C, and (2) stabilizes the covalent protein cofactor linkage up to approximately 70 degrees C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 degrees C of approximately 25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a approximately 20 degrees C higher value on the temperature scale.     

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).     

Photoaffinity labeling of the glucagon receptor with a new glucagon analog

The flash excitation of the pyridoxal 5'-phosphate cofactor of glycogen phosphorylase b by an ultraviolet laser produces a transient state from a proton transfer of the bound cofactor. The rate of decay of this transient state is sensitive to the ionization state of the cofactor. This proved a useful probe for the ionization state of the 5'-phosphate group of the cofactor on the binding by the enzyme of various substrates. The decay rate data show, for the binding of glucose 1-phosphate, a partially negative 5'-HPO4- and evidence for a PO4-PO4 interaction. The data is interpreted in terms of a dynamic shift of substrates at the active site.     

Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors

The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with 31P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH2 group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyridoxal 5'-phosphate as a catalytic and conformational cofactor of muscle glycogen phosphorylase B

This review summarizes data on structure of muscle glycogen phosphorylase b and the role of the cofactor pyridoxal 5"-phosphate in catalysis and stabilizing the native conformation of the enzyme. Specific attention is paid to the stabilizing role of pyridoxal 5"-phosphate upon denaturation of phosphorylase b. Stability of holoenzyme, apoenzyme, and enzyme reduced by sodium borohydride is compared.     

Control of phosphorylase b conformation by a modified cofactor: crystallographic studies on R-state glycogen phosphorylase reconstituted with pyridoxal 5''-diphosphate.

Previous crystallographic studies on glycogen phosphorylase have described the different conformational states of the protein (T and R) that represent the allosteric transition and have shown how the properties of the 5'-phosphate group of the cofactor pyridoxal phosphate are influenced by these conformational states. The present work reports a study on glycogen phosphorylase b (GPb) complexed with a modified cofactor, pyridoxal 5'-diphosphate (PLPP), in place of the natural cofactor. Solution studies (Withers, S.G., Madsen, N.B., & Sykes, B.D., 1982, Biochemistry 21, 6716-6722) have shown that PLPP promotes R-state properties of the enzyme indicating that the cofactor can influence the conformational state of the protein. GPb complexed with pyridoxal 5'-diphosphate (PLPP) has been crystallized in the presence of IMP and ammonium sulfate in the monoclinic R-state crystal form and the structure refined from X-ray data to 2.8 A resolution to a crystallographic R value of 0.21. The global tertiary and quaternary structure in the vicinity of the Ser 14 and the IMP sites are nearly identical to those observed for the R-state GPb-AMP complex. At the catalytic site the second phosphate of PLPP is accommodated with essentially no change in structure from the R-state structure and is involved in interactions with the side chains of two lysine residues (Lys 568 and Lys 574) and the main chain nitrogen of Arg 569. Superposition of the T-state structure shows that were the PLPP to be incorporated into the T-state structure there would be a close contact with the 280s loop (residues 282-285) that would encourage the T to R allosteric transition. The second phosphate of the PLPP occupies a site that is distinct from other dianionic binding sites that have been observed for glucose-1-phosphate and sulfate (in the R state) and for heptulose-2-phosphate (in the T state). The results indicate mobility in the dianion recognition site, and the precise position is dependent on other linkages to the dianion. In the modified cofactor the second phosphate site is constrained by the covalent link to the first phosphate of PLPP. The observed position in the crystal suggests that it is too far from the substrate site to represent a site for catalysis.     

Phosphorylase: control and activity

Recent results from the crystallographic studies on glycogen phosphorylase b at 2 A resolution are reviewed with special reference to other themes of the meeting. The structural similarity of the fold of 150 residues in phosphorylase to the observed in lactate dehydrogenase is discussed and the binding sites for NADH in phosphorylase are described. The binding of the potent inhibitor glucose-1,2-cyclic phosphate to phosphorylase b in the crystal has been studied at 3 A resolution. The results are compared with those previously obtained for glucose-1-phosphate and discussed with reference to proposals for a mechanism of catalysis that involves the essential cofactor pyridoxal phosphate.     

Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.

A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase.     

The glycine-rich region of Escherichia coli D-serine dehydratase. Altered interactions with pyridoxal 5'-phosphate produced by substitution of aspartic acid for glycine

Replacement of glycine by aspartic acid at either of two sites in a conserved, glycine-rich region inactivates the pyridoxal 5'-phosphate-dependent enzyme D-serine dehydratase (DSD) from Escherichia coli. To investigate why aspartic acid at position 279 or 281 causes a loss of activity, we measured the affinity of the G----D variants for pyridoxal 5'-phosphate and a cofactor:substrate analog complex and compared the UV, CD, and fluorescence properties of wild-type D-serine dehydratase and the inactive variants. The two G----D variants DSD(G279D) and DSD (G281D) displayed marked differences from wild-type D-serine dehydratase and from each other with respect to their affinity for pyridoxal 5'-phosphate and for a pyridoxal 5'-phosphate:glycine Schiff base. Compared to the wild-type enzyme, the cofactor affinity of DSD(G279D) and DSD(G281D) was decreased 225- and 50-fold, respectively, and the ability to retain a cofactor:glycine complex was decreased 765- and 1970-fold. The spectral properties of the inactive variants suggest that they form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation. Moreover, the amount of cofactor aldamine in equilibrium with cofactor Schiff base is increased in DSD(G279D) and DSD(G281D) relative to that in wild-type DSD. Collectively, our findings indicate that introduction of a carboxymethyl side chain at G-279 or G-281 directly or indirectly disrupts catalytically essential protein-cofactor and protein-substrate interactions and thereby prevents processing of the enzyme bound cofactor:substrate complex. The conserved glycine-rich region is thus either an integral part of the D-serine dehydratase active site or conformationally linked to it.     

The Fourier transforms of the laser-induced absorption decay from glycogen phosphorylase and DOPA decarboxylase

Fourier analysis of the laser-induced absorption decay curves of 3,4-dihydroxyphenylalanine (DOPA) decarboxylase and glycogen phosphorylase demonstrates a powerful technique in the analysis of complicated decay behavior. Phosphorylase which uses the pyridoxal 5'-phosphate cofactor in an unknown manner exhibits over weak absorption an intense decay while decarboxylase demonstrates only weak absorption. Fourier analysis of the decay curves clearly shows that phosphorylase has an intense absorption decay in the midst of three weaker ones and that decarboxylase only has three weak decays. This conclusion justifies the isolation and use of the intense decay of phosphorylase as an observable in the study of protein dynamics at the active site about the cofactor. The decay has demonstrated a movement of positive charge to substrate in the mechanism of phosphorylation of glycogen units.     

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.     

Studies on the changes in protein fluorescence and enzymic activity of aspartate aminotransferase on binding of pyridoxal 5′-phosphate

1. The alpha and beta subforms of aspartate aminotransferase were purified from pig heart. 2. The alpha subform contained 2mol of pyridoxal 5'-phosphate. The apo-(alpha subform) could be fully reactived by combination with 2mol of cofactor. 3. The protein fluorescence of the apo-(alpha subform) decreased non-linearly with increase in enzyme activity and concentration of bound cofactor. 4. It is concluded that the enzyme activity/mol of bound cofactor is largely independent of the number of cofactors bound to the dimer. 5. The beta subform had approximately half the specific enzyme activity of the alpha subform, and contained an average of one active pyridoxal 5'-phosphate molecule per molecule, which could be removed by glutamate, and another inactive cofactor which could only be removed with NaOH. 6. On recombination with pyridoxal 5'-phosphate the protein fluorescence of the apo-(beta subform) decreased linearly, showing that each dimeric enzyme molecule contained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mechanism for this enzyme.     

Functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate in phosphorylase: a study using pyridoxal-reconstituted enzyme as a model system

Pyridoxal-reconstituted phosphorylase was used as a model system to study the possible functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate (PLP) in rabbit muscle glycogen phosphorylase. Kinetic study was conducted by using competitive inhibitors of phosphite, an activator, and alpha-D-glucopyranose 1-phosphate (glucose-1-P) to study the relationship between the PLP phosphate and the binding of glucose-1-P to phosphorylase. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy of fluorophosphate bound to pyridoxal phosphorylase showed that its ionization state did not change during enzymatic catalysis. Evaluation of the apparent kinetic parameters for the activation of pyridoxal phosphorylase with different analogues having varied pKa2 values demonstrated a dependency of KM on pKa2. Molybdate, capable of binding as chelates in a trigonal-bipyramidal configuration, was tested for its inhibitory property with pyridoxal phosphorylase. On the basis of the results in this study, several conclusions may be drawn: (1) The bound phosphite in pyridoxal phosphorylase and, possibly, the 5'-phosphoryl group of PLP in native phosphorylase do not effect the glucose-1-P binding. (2) One likely function of the 5'-phosphoryl group of PLP in native phosphorylase is acting as an anchoring point to hold the PLP molecule and/or various amino acid side chains in a proper orientation for effective catalysis. (3) The force between the PLP phosphate and its binding site in phosphorylase is mainly electrostatic; a change of ionization state during catalysis is unlikely. (4) Properties of the central atoms of different anions are important for their effects as either activators or inhibitors of pyridoxal phosphorylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The turnover of skeletal muscle glycogen phosphorylase studied using the cofactor, pyridoxal phosphate, as a specific label

The turnover of glycogen phosphorylase has been measured using the cofactor, pyridoxal phosphate, as a label specific for this enzyme in skeletal muscle. Radiolabelled pyridoxine administered in vivo is incorporated into a protein-bound fraction in skeletal muscle, shown by several criteria to be equivalent to glycogen phosphorylase. This pool of radiolabel disapears slowly with a half-life of 11.9 days, taken to be a good estimate of the intracellular half-life of the enzyme. The use of the cofactor in this fashion minimises overestimation of half-life that results from reincorporation of the label. Further, premature dissociation of the cofactor from native enzyme, which would lead to underestimation of half-life, is unlikely. At the level of sensitivity given by this method there was little evidence for the appearance of pyridoxal phosphate-labelled degradation intermediates of the enzyme.     

Purification and characterization of a glycogen phosphorylase analog missing the amino-terminal segment

A glycogen phosphorylase analog missing only the amino-terminal 16 to 18 residues, which include the phosphorylation site, was produced by subtilisin Carlsberg cleavage of phosphorylase b in the presence of caffeine. The analog, named phosphorylase b's, was purified, and its enzymatic properties were compared with those of phosphorylase b. The KM's for glucose 1-phosphate are similar, but phosphorylase b's has a VM 43% higher than that of phosphorylase b. Also, phosphorylase b's is less sensitive to inhibition by glucose 6-phosphate and stimulation by sodium fluoride than is phosphorylase b. The subunit interactions in the two enzyme forms were also compared. The monomer-monomer interactions in phosphorylase b's are weaker than in phosphorylase b, as evidenced by a faster rate of resolution of the coenzyme, pyridoxal phosphate, from phosphorylase b's. The dimer-dimer interactions are also weaker in phosphorylase b's than in phosphorylase b, because phosphorylase b's does not form tetramers or crystals as readily as does phosphorylase b. Because removal of the amino-terminal segment changes the properties of the enzyme, this segment must be interacting with other parts of the protein. This statement conflicts with previous interpretation of X-ray crystallographic data that suggest that the amino-terminal region of phosphorylase b is freely mobile. Possible explanations for this contradiction are discussed.     

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.     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Effect of denervation on the expression of glycogen phosphorylase in mouse skeletal muscle.

After sciatectomy of the left hind-limb of C57BL/J mice, a denervation-induced muscular atrophy ensued and was accompanied by a decrease in the specific activity of glycogen phosphorylase to approx. 25% of control values. The cofactor of phosphorylase, pyridoxal 5'-phosphate, was used as a specific label in the determination of the degradation rate of the enzyme following nerve section. After a delay of 3-4 days, phosphorylase was degraded approx, twice as rapidly in the denervated gastrocnemius (0.20 day-1) as in the control muscle (0.12 day-1). The effect of denervation on phosphorylase mRNA was measured by quantitative Northern-blot analysis using a rat skeletal-muscle phosphorylase cDNA probe. After an initial rapid decline, phosphorylase mRNA levels stabilized in denervated muscle at 50% of the value measured in the contralateral control muscle.