Conformational changes of spin-labeled native and modified phosphorylase B

The phosphorylase B labelled with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-iodacetamide (phosphorylase I) and with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-ethylmaleinimide (phosphorylase II) was studied. It was shown that label I is characterized by a greater mobility with respect to the protein as compared to label II. In spin-labelled preparations of phosphorylase B the 1,5--2,0 SH-groups of the enzyme monomer having no effect on the enzyme activity were modified. The effects of AMP, glucose-1-phosphate and glucose-6-phosphate on the EPR spectrum of phosphorylase I were studied. The greatest changes in the spectrum (especially in the high field line) were found to occur in the presence of glucose-6-phosphate. These changes are due to the increase in the degree of anisotropic spin rotation. The experimental and theoretical spectra allowing to determine the correlation time for the protein moiety (tau b = 160 ns) were shown to be similar. The local conformation changes were found to occur in the vicinity of one of the two label-bound SH-groups of phosphorylase I. The EPR spectra demonstrate the S-shaped dependence of mobility of phosphorylase I label on concentration of glucose-6-phosphate (0,1--10 mM). In the presence of AMP no S-shaped dependence is observed. Reduced NaBH4 phosphorylase I does not reveal the S-shaped dependence of the label mobility on concentration of glucose-6-phosphate. The degree of the label immobilization in the apo-phosphorylase I--pyridoxal-5-chloromethylphosphonate complex in the presence of glucose-6-phosphate and AMP is the same as in cholophosphorylase I; however, in contrast to the choloenzyme it does not depend on glucose-6-phosphate (0,1--10,0 mM). The changes in the mobility of the spin label of apophosphorylase I and its complex with the AMP analog--adenosine-5'-chloromethylphosphonate--during the choloenzyme reconstruction by pyridoxalphosphate are indicative of participation of AMP and the phosphate group of AMP in the formation of the enzyme active center.     

Heterotropic interactions of ligands with phosphorylase b.

1. The interaction of rabbit muscle glycogen phosphorylase b with pairs of ligands has been examined. 2. The electron spin resonance spectrum of a spin label, covalently attached to the protein, provided information about dissociation constants, formation of ternary complexes and both negative and positive interactions between different ligand pairs. 3. AMP competes with a series of nucleotides (ADP, ATP, CMP aand cytosine) but with adenosine a ternary enzyme - AMP - adenosine complex can be formed. 4. ADP binding is tight and ADP inhibits the AMP activation of phosphorylase b in a physiologically important concentration range. 5. The substrates glucose 1-phosphate and glycogen tighten AMP binding in the ternary complex as does the competitive inhibitor UDPG. Inorganic phosphate is different in this respect. Gluconolactone, a transition state analogue, competes with glucose 1-phosphate (but not with glycogen) but does not prevent completely the binding of the sugar phosphate. 6. The effect of glucose b-phosphate on phosphorylase is rather complex as it 'formally competes' with both AMP and UDPG probably mediated by a conformational changes and not by 'direct' interactions with these two ligands. Glycerol 2-phosphate, a commonly used buffer for phosphorylase, also shows complex interactions.     

Conformational changes in glycogen phosphorylase studied with a spin-label probe.

Phosphorylase b and a were covalently modified on essentially one -- SH group per subunit by a spin label 4-(2-iodoacetamido)2,2,6,6-tetramethyl piperidinyloxyl. The labelled enzyme is fully active and exhibits all the characteristics of the native molecule. The electron spin resonance spectrum of the label depends on the nature of the ligand that is bound to the enzyme. This property of the spin label is used to study the interaction between the enzyme (both in the b and a forms) and activators (AMP, IMP, CMP), inhibitors (ADP, ATP, UDPG, glucose 6-phosphate), substrates (phosphate and glucose 1-phosphate) and other ligands (adenosine, beta-glycerol-2-phosphate). The interactions are analysed in terms of the apparent ligand dissociation constants and the multiplicity of conformations that this regulatory enzyme exhibits.     

Some properties of starch phosphorylase from cotyledons of germinating seeds of Voandzeia subterranea.

Two isoenzymes (Forms I and II) of starch phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) were found in cotyledons of germinating seeds of Voandzeia subterranea L. Thouars. Phosphorylase I, which was the major component, had a pH optimum of 5.5--5.6, whereas phosphorylase II had a pH optimum of 6.1--6.3. Phosphorylase I had a molecular weight of 204 000 +/- 4000 and a subunit molecular weight of about 95 000. Phosphorylase I was stimulated by Mg2+, Mn2+, AMP, cyclic AMP, pyruvate and EDTA, but inhibited by Fe2+, Cu2+, Zn2+ and ATP. Stimulation of phosphorulase I by AMP was accompanied by changes in the affinity of the enzyme for glucose-1-phosphate in the presence of increasing AMP concentrations, and of AMP in the presence of increasing glucose-1-phosphate concentrations. Double-reciprocal plots of initial velocity data were non-linear (convex up) at low glucose-1-phosphate concentrations but became linear in the presence of AMP or ATP. Double-reciprocal plots were linear at high glucose-1-phosphate concentrations in the absence or presence of modifiers.     

Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site

Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.     

Characteristics of the dephosphorylated form of phosphorylase purified from rat liver and measurement of its activity in crude liver preparations.

The phosphorylated form of liver glycogen phosphorylase (alpha-1,4-glucan : orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) (phosphorylase a) is active and easily measured while the dephosphorylated form (phosphorylase b), in contrast to the muscle enzyme, has been reported to be essentially inactive even in the presence of AMP. We have purified both forms of phosphorylase from rat liver and studied the characteristics of each. Phosphorylase b activity can be measured with our assay conditions. The phosphorylase b we obtained was stimulated by high concentrations of sulfate, and was a substrate for muscle phosphorylase kinase whereas phosphorylase a was inhibited by sulfate, and was a substrate for liver phosphorylase phosphatase. Substrate binding to phosphorylase b was poor (KM glycogen = 2.5 mM, glucose-1-P = 250 mM) compared to phosphorylase a (KM glycogen = 1.8 mM, KM glucose-1-P = 0.7 mM). Liver phosphorylase b was active in the absence of AMP. However, AMP lowered the KM for glucose-1-P to 80 mM for purified phosphorylase b and to 60 mM for the enzyme in crude extract (Ka = 0.5 mM). Using appropriate substrate, buffer and AMP concentrations, assay conditions have been developed which allow determination of phosphorylase a and 90% of the phosphorylase b activity in liver extracts. Interconversion of the two forms can be demonstrated in vivo (under acute stimulation) and in vitro with little change in total activity. A decrease in total phosphorylase activity has been observed after prolonged starvation and in diabetes.     

Effect of polymyxins on glycogen phosphorylase

AMP-dependent activity of glycogen phosphorylase b is stimulated by the polymyxins A, B, D, and E. Kinetic studies indicate that these cyclic peptide antibiotics at low concentrations greatly enhance AMP-activation of the enzyme. The presence of polymyxins in the assay system leads to (a) partial desensitization of allosteric interactions toward AMP, (b) lowering of K_m for the substrates glucose-1-phosphate and glycogen, and (c) reversal of the glucose-6-phosphate inhibition. in contrast to phosphorylase b, neither AMP-phosphorylase b′ system nor phosphorylase a (with or without AMP) is considerably activated by polymyxins.     

Multiple forms of synthase D phosphatase and phosphorylase a phosphatase in liver and regulatory effects of metabolites on their activities

The smooth endoplasmic reticulum (ER) and cytosol fractions of liver homogenates exhibit phosphoprotein phosphatase activity towards glycogen synthase D and phosphorylase a. The following observations suggest that liver contains multiple forms of these phosphatases. Synthase phosphatase activity in either fraction was more readily inactivated by heating than phosphorylase phosphatase activity. Both synthase phosphatase and phosphorylase phosphatase activities in smooth ER were non-competitively inhibited by Mg2+, but were activated by this ion in the cytosol. Synthase phosphatase activities in cytosol and smooth ER were stimulated by a number of sugar phosphates, particularly glucose-1-phosphate, galactose-6-phosphate and fructose-6-phosphate. Erythrose-4-phosphate stimulated synthase phosphatase activity in the cytosol, but inhibited the microsomal enzyme. Phosphorylase phosphatase activities in either fraction were inhibited by most sugar phosphates. Adenosine mono-, di- and tri-phosphates inhibited phosphatase activities in both fractions. Low concentrations of AMP and ADP inhibited phosphorylase phosphatase activities to a greater extent than synthase phosphatase activities. Chromatography of the smooth ER fraction on DEAE-cellulose resulted in the separation of synthase phosphatase from phosphorylase phosphatase, as soluble proteins. The elution profile for the microsomal phosphatase was different from that for the cytosol enzymes. It is concluded that: both synthase phosphatase and phosphorylase phosphatase in liver have at least two isoenzyme forms; synthase phosphatase and phosphorylase phosphatase are separate enzymes; the different behaviour of microsomal and cytosol phosphatases towards divalent cations and sugar phosphates provides a potential mechanism for the differential regulation of these activities in liver.     

Catalysis in the crystal: synchrotron radiation studies with glycogen phosphorylase b.

Direct observation of the progress of a catalysed reaction in crystals of glycogen phosphorylase b has been made possible through fast crystallographic data collection achieved at the Synchrotron Radiation source at Daresbury, UK. In the best experiments, data to 2.7 A resolution (some 108,300 measurements; 21,200 unique reflections) were measured in 25 min. In a series of time-resolved studies in which the control properties of the enzyme were exploited in order to slow down the reaction, the conversion of heptenitol to heptulose-2-phosphate, the phosphorylysis of maltoheptaose to yield glucose-1-phosphate and the oligosaccharide synthesis reaction involving maltotriose and glucose-1-phosphate have been monitored in the crystal. Changes in electron density in the difference Fourier maps are observed as the reaction proceeds not only at the catalytic site but also the allosteric and glycogen storage sites. Phosphorylase b is present in the crystals in the T state and under these conditions exhibits low affinity for both phosphate and oligosaccharide substrates. There are pronounced conformational changes associated with the formation and binding of the high-affinity dead-end product, heptulose-2-phosphate, which show that movement of an arginine residue, Arg 569, is critical for formation of the substrate-phosphate recognition site. The results are discussed with reference to proposals for the enzymic mechanism of phosphorylase. The feasibility for time-resolved studies on other systems and recent advances in this area utilizing Laue diffraction are also discussed.     

The amino-terminal tail of glycogen phosphorylase is a switch for controlling phosphorylase conformation, activation, and response to ligands

Glycogen phosphorylase is a muscle enzyme which metabolizes glycogen, producing glucose-1-phosphate, which can be used for the production of ATP. Phosphorylase activity is regulated by phosphorylation/dephosphorylation, and by the allosteric binding of numerous effectors. In this work, we have studied 10 site-directed mutants of glycogen phosphorylase (GP) in its amino-terminal regulatory region to characterize any changes that the mutations may have made on its structure or function. All of the GP mutants had normal levels of activity in the presence of the allosteric activator AMP. Some of the mutants were observed to have altered AMP-binding characteristics, however. R16A and R16E were activated at very low AMP concentration and crystallized at low temperature, like the phosphorylated form of GP, phosphorylase a, and unlike the dephospho-form, phosphorylase b. This indicates that even without phosphorylation, the structures of these mutants are more like phosphorylase a than phosphorylase b. These mutants were also very poorly phosphorylated in the presence of the inhibitor glucose, while phosphorylase b was phosphorylated normally with this inhibitor present. In contrast to R16A and R16E, four other mutants behaved like phosphorylase b after phosphorylation. R69E was only partially activated by phosphorylation, and I13G, R43E, and R43E/R69E were completely inactive after phosphorylation. We propose a model for the many functions of the amino terminus to explain the many varied effects of these mutations.     

Inhibition of rabbit muscle glycogen phosphorylase by D-gluconohydroximo-1,5-lactone-N-phenylurethane

The effect of the beta-glycosidase inhibitor D-gluconohydroximo-1,5-lactone-N-phenylurethane (PUG) on the kinetic and ultracentrifugation properties of glycogen phosphorylase has been studied. Recent crystallographic work at 2.4 A resolution [D. Barford et al. (1988) Biochemistry 27, 6733-6741] has shown that PUG binds in the catalytic site of phosphorylase b crystals with its gluconohydroximolactone moiety occupying a position similar to that observed for other glucosyl compounds and the N-phenylurethane side chain fitting into an adjacent cavity with little conformational change in the enzyme. In solution, PUG was shown to be a potent inhibitor of phosphorylase b, directly competitive with alpha-D-glucopyranose 1-phosphate (glucose-1-P) (Ki = 0.40 mM) and noncompetitive with respect to glycogen and AMP. When PUG was tested for synergistic inhibition in the presence of caffeine, the Dixon plots of reciprocal velocity versus PUG concentration at different fixed caffeine concentrations provided intersecting lines with interaction constant (alpha) values of 0.95-1.38, indicating that the binding of one inhibitor is not significantly affected by the binding of the other. For glycogen phosphorolysis, PUG was noncompetitive with respect to phosphate, suggesting that it can bind to the central enzyme-AMP-glycogen-phosphate complex. PUG was shown to inhibit phosphorylase alpha (without AMP) activity (Ki = 0.43 mM) in a manner similar to that of the b form. However, in the presence of AMP, PUG exhibited complex kinetics, acting as a noncompetitive inhibitor with respect to glucose-1-P, while a twofold decrease of PUG binding to the enzyme-AMP-glycogen complex was observed. Ultracentrifugation experiments demonstrated that PUG does not cause any significant dissociation of phosphorylase alpha tetramer. Furthermore the dimerization of phosphorylase alpha by glucose is completely prevented in the presence of PUG. These observations are consistent with PUG binding to both the R and the T conformations of phosphorylase.     

An engineered liver glycogen phosphorylase with AMP allosteric activation

Liver and muscle glycogen phosphorylases, which are products of distinct genes, are both activated by covalent phosphorylation, but in the unphosphorylated (b) state, only the muscle isozyme is efficiently activated by the allosteric activator AMP. The different responsiveness of the phosphorylase isozymes to allosteric ligands is important for the maintenance of tissue and whole body glucose homeostasis. In an attempt to understand the structural determinants of differential sensitivity of the muscle and liver isozymes to AMP, we have developed a bacterial expression system for the liver enzyme, allowing native and engineered proteins to be expressed and characterized. Engineering of the single amino acid substitutions Thr48Pro, Met197Thr and the double mutant Thr48Pro, Met197Thr in liver phosphorylase, and Pro48Thr in muscle phosphorylase, did not qualitatively change the response of the two isozymes to AMP. These sites had previously been implicated in the configuration of the AMP binding site. However, when nine amino acids among the first 48 in liver phosphorylase were replaced with the corresponding muscle phosphorylase residues (L1M2-48L49-846), the engineered liver enzyme was activated by AMP to a higher maximal activity than native liver phosphorylase. Interestingly, the homotropic cooperativity of AMP binding was unchanged in the engineered phosphorylase b protein, and heterotropic cooperativity between the glucose-1-phosphate and AMP sites was only slightly enhanced. The native liver, native muscle and L1M2-48L49-846 phosphorylases were converted to the a form by treatment with purified phosphorylase kinase; the maximal activity of the chimeric a enzyme was greater than the native liver a enzyme and approached that of muscle phosphorylase a. From these results we suggest that tissue-specific phosphorylase isozymes have evolved a complex mechanism in which the N-terminal 48 amino acids modulate intrinsic activity (Vmax), probably by affecting subunit interactions, and other, as yet undefined regions specify the allosteric interactions with ligands and substrates.     

Modeling the biochemical differences between rabbit muscle and human liver phosphorylase

Glycogen phosphorylases catalyze the regulated breakdown of glycogen to glucose-1-phosphate. In mammals, glycogen phosphorylase occurs in three different isozymes called liver, muscle, and brain after the tissues in which they are preferentially expressed. The muscle isozyme binds and is activated cooperatively by AMP. In contrast, the liver enzyme binds AMP noncooperatively and is poorly activated. The amino acid sequence of human liver phosphorylase is 80% identical with rabbit muscle phosphorylase, and those residues which contact AMP are conserved. Using computer graphics software, we replaced side chains of the known rabbit muscle structure with those of human liver phosphorylase and interpreted the effects of these changes in order to account for the biochemical differences between them. We have identified two substitutions in liver phosphorylase potentially important in altering the cooperative binding and activation of this isozyme by AMP.     

Combined kinetic mechanism describing activation and inhibition of muscle glycogen phosphorylase b by adenosine 5'-monophosphate

The kinetic analysis of the glycogen chain growth reaction catalyzed by glycogen phosphorylase b from rabbit skeletal muscle has been carried out over a wide range of concentrations of AMP under the saturation of the enzyme by glycogen. The applicability of 23 different variants of the kinetic model involving the interaction of AMP and glucose 1-phosphate binding sites in the dimeric enzyme molecule is considered. A kinetic model has been proposed which assumes: (i) the independent binding of one molecule of glucose 1-phosphate in the catalytic site on the one hand, and AMP in both allosteric effector sites and both nucleoside inhibitor sites of the dimeric enzyme molecule bound by glycogen on the other hand; (ii) the binding of AMP in one of the allosteric effector sites results in an increase in the affinity of other allosteric effector site to AMP; (iii) the independent binding of AMP to the nucleoside inhibitor sites of the dimeric enzyme molecule; (iv) the exclusive binding of the second molecule of glucose 1-phosphate in the catalytic site of glycogen phosphorylase b containing two molecules of AMP occupying both allosteric effector sites; and (v) the catalytic act occurs exclusively in the complex of the enzyme with glycogen, two molecules of AMP occupying both allosteric effector sites, and two molecules of glucose 1-phosphate occupying both catalytic sites.     

The interplay between covalent and non-covalent regulation of glycogen phosphorylase. The role of different effectors of phosphorylase b on the phosphorylase b to a conversion rate.

Glycogen phosphorylase b is converted to glycogen phosphorylase a, the covalently activated form of the enzyme, by phosphorylase kinase. Glc-6-P, which is an allosteric inhibitor of phosphorylase b, and glycogen, which is a substrate of this enzyme, are already known to have respectively an inhibiting and activating effect upon the rate of conversion from phosphorylase b to phosphorylase a by phosphorylase kinase. In the former case, this effect is due to the binding of glucose-6-phosphate to glycogen phosphorylase b. In order to investigate whether or not the rate of conversion of glycogen phosphorylase b to phosphorylase a depends on the conformational state of the b substrate, we have tested the action of the most specific effectors of glycogen phosphorylase b activity upon the rate of conversion from phosphorylase b to phosphorylase a at 0 degrees C and 22 degrees C : AMP and other strong activators, IMP and weak activators, Glc-6-P, glycogen. Glc-1-P and phosphate. AMP and strong activators have a very important inhibitory effect at low temperature, but not at room temperature, whereas the weak activators have always a very weak, if even existing, inhibitory effect at both temperatures. We confirmed the very strong inhibiting effect of Glc-6-P at both temperatures, and the strong activating effect of glycogen. We have shown that phosphate has a very strong inhibitory effect, whereas Glc-1-P has an activating effect only at room temperature and at non-physiological concentrations. The concomitant effects of substrates and nucleotides have also been studied. The observed effects of all these ligands may be either direct ones on phosphorylase kinase, or indirect ones, the ligand modifying the conformation of phosphorylase b and its interaction with phosphorylase kinase. Since we have no control experiments with a peptidic fragment of phosphorylase b, the interpretation of our results remains putative. However, the differential effects observed with different nucleotides are in agreement with the simple conformational scheme proposed earlier. Therefore, it is suggested that phosphorylase kinase recognizes differently the different conformations of glycogen phosphorylase b. In agreement with such an explanation, it is shown that the inhibiting effect of AMP is mediated by a slow isomerisation which has been previously ascribed to a quaternary conformational change of glycogen phosphorylase b. The results presented here (in particular, the important effect of glycogen and phosphate) are also discussed in correlation with the physiological role of the different ligands as regulatory signals in the in vivo situation where phosphorylase is inserted into the glycogen particle.     

Conformational changes associated with transient activation of phosphorylase in glycogen particles. Studies using activity, electron-spin-resonance and phosphorus-nuclear-magnetic-resonance measurements.

1. Calcium-dependent transient phosphorylation of phorphorylase b has been monitored in a rabbit muscle glycogen particle fraction. Using a phosphorus nuclear magnetic resonance assay, the changes in concentrations of small phosphate-containing metabolites associated with this event have been measured. In addition, the conformation of phosphorylase has been monitored during transient activation by observing changes in the electron spin resonance signal from added spin-labelled phosphorylase. 2. The transient activation was associated with a loss of glucose-6-phosphate from phosphorylase b; newly formed phosphorylase a binds the nucleotides ADP, AMP, or IMP. Because of the fast interconversion of these nucleotides the species bound to phosphorylase a change throughout the process. 3. Lowering the [Mg2+] : [Ca2+] ratio during transient activation causes accumulation of ADP. Electron spin resonance data from spin-labelled phosphorylase shows that, under these conditions, ADP binding to phosphorylase a is potentiated. 4. Calcium-dependent activation in the glycogen particle fraction is compared to the activation of phosphorylase in vivo.     

Effect of metabolites and phosphorylase on the D to I conversion of glycogen synthase from human polymorphonuclear leukocytes.

The D to I conversion of glycogen synthase from human polymorphonuclear leukocytes was examined both in a gel-filtered homogenate and in a preparation of glycogen particles with adhering enzymes, purified by chromatography on concanavalin A bound to Sepharose. It was found that glucose 6-phosphate as well as mannose 6-phosphate, glucosamine 6-phosphate, and 2-deoxy-glucose 6-phosphate activated the reaction, whereas the corresponding sugars were without effect. Mn2+ and Ca2+ increased the conversion rate by 51% and 27%, respectively, whereas Mg2+ and inorganic phosphate were without effect. Sodium fluoride inhibited the reaction completely. Glycogen inhibited the reaction in physiological concentrations and 0.5 mM glucose 6-phosphate was able to overcome this inhibition. MgATP greatly augmented the inhibition caused by glycogen in the glycogen particle preparation. This combined effect could be overcome by glucose 6-phosphate in concentrations from 0.1 to 1 mM. Phosphorylase alpha purified from human polymorphonuclear leukocytes inhibited the D to I conversion in a glycogen particle preparation. The inhibition was counteracted by glucose 6-phosphate and to a lesser degree by AMP. Phosphorylase beta was also inhibitory, but only at higher concentrations than phosphorylase alpha. No phosphorylase phosphatase activity was found in the glycogen particle preparation, which may indicate that chromatography on concanavalin A-Sepharose separates this enzyme from the synthase phosphatase or partially destroys the activity of a hypothetical common protein phosphatase.     

Interaction of glycogen phosphorylase with 8-azidoadenosine 5'-monophosphate, a photoaffinity analog of AMP

The ability of 8-azidoadenosine 5'-monophosphate (N3AMP) to act as a photoaffinity label for the AMP binding site on glycogen phosphorylase (EC 2.4.1.1) was tested. 8-Azidoadenosine 5'-monophosphate can replace AMP as an allosteric modifier of both phosphorylases a and b; the pH optimum and the extent of activation are comparable to that observed with AMP. 8-Azidoadenosine 5'-monophosphate resembles the natural activator in having a higher affinity for phosphorylase a. The effects of 8-azidoadenosine 5'-monophosphate and AMP on phosphorylase b are additive when each is present at a concentration which gives less than 50% activation. Increasing the concentration of the substrate, glucose 1-phosphate, decreases the apparent activation constant (Ka) for the interaction of 8-azidoadenosine 5'-monophosphate with phosphorylase b. Glucose 6-phosphate is an inhibitor of phosphorylase b with either AMP or 8-azidoadenosine 5'-monophosphate. In the presence of ultraviolet light, 8-azidoadenosine 5'-monophosphate is irreversibly incorporated into phosphorylase a; incorporation at the allosteric site can be reduced if AMP is added prior to irradiation. Under the conditions used in the photolysis experiments, 3--5% of the available AMP sites were labeled with 8-azidoadenosine 5'-monophosphate. The data indicate the potential usefulness of 8-azidoadenosine 5'-monophosphate as a probe for the AMP site on phosphorylase.     

Interaction of phosphorylase b with eosin. Influence of substrate and effectors on eosin-enzyme complexes.

The interactions of rabbit muscle glycogen phosphorylase b with Eosin (2',4',5',7'-tetrabromofluorescein) was studied. Eosin was found to be an effective inhibitor of the enzyme. The inhibition constants for the dye were estimated to be approx. 36 and 60 microM with respect to AMP and glucose 1-phosphate respectively. The binding of Eosin to phosphorylase b is accompanied by a red-shift of about 12 nm in the dye absorption-spectrum maximum, indicating low-polarity binding sites on the enzyme molecule for the dye. The absorbance in the difference absorption maximum at 537 nm was utilized to follow the conjugation of phosphorylase b with Eosin. Scatchard plots of the titration data revealed the existence of at least two classes of binding sites on the protein molecule for Eosin, and the dissociation constants measured in Tris/HCl buffer, pH 7.0 (IO.091), were 7.7 and 41.7 microM respectively. The influence of the substrates and effectors on Eosin-enzymes complexes was used to study the ligand-phosphorylase b interactions. IMP displaced the dye completely from the enzyme, indicating that there are two IMP-binding sites per phosphorylase b monomer. AMP binding to the enzyme with respect to Eosin concentration is of two types: a non-competitive one for the high-affinity site for AMP and a competitive one for the low-affinity site for the activator. The effects of glucose 6-phosphate, ATP, Pi and glycerol 2-phosphate in the system are in according dance with a partially competitive model. Glucoes 1-phosphate and UDP-glucose appear to affect only the high-affinity site for Eosin, whereas glucose and glycogen have no effect on Eosin-phosphorylase b complexes. Our results suggest that Eosin can be used as an efficient optical probe for studying the phosphorylase b system.     

Regulation of glycogen phosphorylase. Role of the peptide region surrounding the phosphoserine residue in determining enzyme properties.

A phosphopeptide which contains 14 residues including phosphoserine and which is derived from the NH2-terminal region of skeletal muscle glycogen phosphorylase (Nolan, C., Novoa, W. B., Krebs, E. G., and Fischer, E. H. (1964) Biochemistry 3, 542-551) has been shown to induce the enzymic properties of phosphorylase a in phosphorylase b and b'. When phosphorylase b is incubated with the phosphorylated tetradecapeptide, the following changes occur: (1) the enzyme becomes partially catalytically active in the absence of AMP; (2) the allosteric interactions of the enzyme are altered, as evidenced by the fact that phosphorylase b does not bind AMP cooperatively, and is no longer inhibited by glucose-6-P; and (3) the enzyme, normally present as a dimer, associates to a tetramer. Phosphorylase b' is a modified form of phosphorylase in which the phosphorylation site has been removed by limited tryptic attack. In the presence of phosphopeptide, 86% of the total enzyme activity can be induced in the absence of AMP. The properties of phosphorylases b and b' with phosphopeptide, cited above, are all characteristics of the phosphonenzyme, phosphorylase a. In addition, evidence is presented that these effects are specific. They are not the result of the polycationic nature of the peptide since they cannot be duplicated by spermine, and the phosphate group must also be present for the peptide to effect changes on the enzyme.