Interaction of flavin mononucleotide with dimeric and tetrameric forms of muscle phosphorylase beta.

Interaction of flavin mononucleotide (FMN) with dimeric and tetrameric forms of rabbit muscle glycogen phosphorylase beta has been studied under the conditions when allosteric activator binding sites are saturated by AMP (1 mM AMP; pH 6.8; 17 degrees C). Simultaneous use of schlieren optical system and photoelectric scanning absorption optical system of analytical ultracentrifuge Spinco, model E, makes it possible to register the oligomeric state of the enzyme and calculate the degree of saturation of individual oligomeric enzyme forms by FMN. The apparent association constant for the equilibrium dimer in equilibrium with tetramer decreased with increasing FMN concentration. The microscopic dissociation constants for the complexes of dimeric and tetrameric forms of glycogen phosphorylase beta with FMN have been found to be equal to 10 and 79 microM, respectively.     

The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation.

The allosteric transition of glycogen phosphorylase promoted by protein phosphorylation is accompanied by the association of a pair of functional dimers to form a tetramer. The conformational changes within the dimer that lead to the creation of a protein recognition surface have been analyzed from a comparison of the crystal structures of T-state dimeric phosphorylase b and R-state tetrameric phosphorylase a. Regions of the structure that participate in the tetramer interface are situated within structural subdomains. These include the glycogen storage subdomain, the C-terminal subdomain and the tower helix. The subdomains undergo concerted conformational transitions on conversion from the T to the R state (overall r.m.s. shifts between 1 and 1.7 A) and, together with the quaternary conformational change within the functional dimer, create the tetramer interface. The glycogen storage subdomain and the C-terminal subdomain are distinct from those regions that contribute to the dimer interface, but shifts in the subdomains are correlated with the allosteric transitions that are mediated by the dimer interface. The structural properties of the tetramer interface are atypical of an oligomeric protein interface and are more similar to protein recognition surfaces observed in protease inhibitors and antibody-protein antigen complexes. There is a preponderance of polar and charged residues at the tetramer interface and a high number of H-bonds per surface area (one H-bond per 130 A2). In addition, the surface area made inaccessible at the interface is relatively small (1,142 A2 per subunit on dimer to tetramer association compared with 2,217 A2 per subunit on monomer-to-dimer association).     

Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b.

The suitability of sedimentation equilibrium for characterizing the self-association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mM Hepes buffer (pH 6.8) supplemented with 1 mM AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as well as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding by trimethylamine N-oxide on the self-association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self-association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer-tetramer equilibrium are being countered by those displacing the T<==>R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme.     

Subunit dissociation in the allosteric regulation of Glycerol kinase from Escherichia coli. 3. Role in desensitization.

The mechanism of desensitization of glycerol kinase to allosteric inhibition by fructose 1,6-bisphosphate caused by salt, urea, and high pH has been examined in the light of the model proposed in an earlier paper [de Riel, J. K., and Paulus H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding. KCl (0.4 M) causes a tenfold decrease in the affinity of tetrameric glycerol kinase for fructose, 1,6-bisphosphate but has no significant effect on the dissociation process itself. Urea (2 M) causes a large increase in the equilibrium constant for the dissociation of the glycerol kinase tetramer to dimer but has no effect on the affinity of the tetramer for the allosteric inhibitor. High pH (9--10) has only a small effect on the subunit dissociation constant but greatly reduces the rates of subunit association and dissociation. Desensitization of glycerol kinase to allosteric inhibition can thus occur by three different mechanisms, two of which are directly related to the polysteric nature of the enzyme.     

Kinetic study on the dimer-tetramer interconversion of phosphorylase b by a stopped-flow X-ray scattering method

The dimer-tetramer interconversion of phosphorylase b induced by the binding of AMP and Mg2+ was monitored using a stopped-flow X-ray scattering method. The rate constants of this second-order reaction have been determined by a nonlinear least-squares method. Burst phases in both radii of gyration and zero-angle intensities were detected at the initial step of the reaction. This suggests that rapid association might take place, followed by a slow association process of which the kinetics were measured in the present study. The radius of gyration of tetrameric phosphorylase b was determined and found to be in excellent agreement with that of phosphorylase a, but different from that of phosphorylase b reported elsewhere (G. Puchwein, O. Kratky, C. F. Golker and E. Helmreich, Biochemistry 9 (1970) 4691). The reason for this inconsistency is discussed.     

D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms

Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit. Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer. A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms. In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer. The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions. Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+. At coenzyme concentrations below half-saturating a monomer is more active than a tetramer. This difference disappears at saturating concentrations of NAD+. Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis. The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis. This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated. Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of muscle glycogen phosphorylase B with flavin mononucleotide and its analogs

The inhibition of rabbit skeletal muscle glycogen phosphorylase B by FMN and its analogues with substituents in the positions 6 and 8 has been studied. Inhibiting action of FMN is manifested in reducing the limiting rate of enzymic reaction and in increasing the half-saturation concentration of AMP. The inhibitor half-saturation values (in microM) increase in the following order: FMN (13,5), 6-bromo-FMN (27), 8 alpha-hydroxy-FMN (30), 8-dimethylamino(nor)-FMN (33), 6-(N-acetyl-L-cysteine-S-yl)-FMN (44), 6-amino-FMN (96), 8-hydroxy(nor)-FMN (109), 6-nitro-FMN (170), 8 alpha-(N-acetyl-L-cysteine-S-yl)-FMN (260). The existence of the glycogen phosphorylase B complexes with FMN or its analogues has been proved by spectrophotometry and sedimentation in analytical ultracentrifuge. FMN has been shown to hinder AMP-induced transition of dimeric form of the enzyme to tetrameric one. AMP at high concentrations has been found to inhibit glycogen phosphorylase B.     

Insights into the structural basis of the pH-dependent dimer-tetramer equilibrium through crystallographic analysis of recombinant Diocleinae lectins

The structural ground underlying the pH-dependency of the dimer-tetramer transition of Diocleinae lectins was investigated by equilibrium sedimentation and X-ray crystal structure determination of wild-type and site-directed mutants of recombinant lectins. Synthetic genes coding for the full-length alpha-chains of the seed lectins of Dioclea guianensis (termed r-alphaDguia) and Dioclea grandiflora (termed r-alphaDGL) were designed and expressed in Escherichia coli. This pioneering approach, which will be described in detail in the present paper, yielded recombinant lectins displaying carbohydrate-binding activity, dimer-tetramer equilibria and crystal structures indistinguishable from their natural homologues. Conversion of the pH-stable tetrameric r-alphaDGL into a structure exhibiting pH-dependent dimer-tetramer transition was accomplished through mutations that abolished the interdimeric interactions at the central cavity of the tetrameric lectins. Both the central and the peripheral interacting regions bear structural information for formation of the canonical legume lectin tetramer. We hypothesize that the strength of the ionic contacts at these sites may be modulated by the pH, leading to dissociation of those lectin structures that are not locked into a pH-stable tetramer through interdimeric contacts networking the central cavity loops.     

Aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12. Concentration-dependent dissociation to dimers in the presence of L-threonine.

The concentration-dependent association-dissociation equilibrium of the bifunctional enzyme aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12 has been investigated at pH 7.6 in the presence of 10 mM L-threonine and 0.1 M KCl by equilibrium gel permeation monitored by a single-photon counting spectrophotometer. The results obtained are consistent with the existence of a dimer-tetramer equilibrium with the association constant of 2.6 X 10(7) M-1 (deltaG0 = -9.9 kcal/mol of dimer). The limiting partition cross-sections estimated by a three-parameter least squares minimization procedure indicate that the molecular radii of the dimer and tetramer are 53.8 A and 70 A, respectively. Both the dimeric and tetrameric forms of the enzyme possess dehydrogenase activity. Treatment of the enzyme with the chaotropic salts, potassium thiocyanate or potassium trichloroacetate, generates a monomeric form that is devoid of dehydrogenase activity. The catalytically inactive monomeric form of the enzyme has a molecular radius between 43 and 45.5 A and a molecular weight of approximately 80,000 as determined by small zone gel chromatography and sedimentation equilibrium studies.     

Regulation of the aggregation state of maize phosphoenolpyruvate carboxylase: evidence from dynamic light-scattering measurements

The molecular weights of different aggregational states of phosphoenolpyruvate carboxylase purified from the leaves of Zea mays have been determined by measurement of the molecular diameter using a Malvern dynamic light scattering spectrometer. Using these data to identify the monomer, dimer, tetramer, and larger aggregate(s) the effect of pH and various ligands on the aggregational equilibria of this enzyme have been determined. At neutral pH the enzyme favored the tetrameric form. At both low and high pH the tetramer dissociated, followed by aggregation to a "large" inactive form. The order of dissociation at least at low pH appeared to be two-step: from tetramer to dimers followed by dimer to monomers. The monomers then aggregate to a large aggregate, which is inactive. The presence of EDTA at pH 8 protected the enzyme against both inactivation and large aggregate formation. Dilution of the enzyme at pH 7 at room temperature results in driving the equilibrium from tetramer to dimer. The presence of malate with EDTA stabilizes the dimer as the predominant form at low protein concentrations. The presence of the substrate phosphoenolpyruvate alone and with magnesium and bicarbonate induced formation of the tetramer, and decreased the dissociation constant (Kd) of the tetrameric form. The inhibitor malate, however, induced dissociation of the tetramer as evidenced by an increase in the Kd of the tetramer.     

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.     

Immobilized active monomers of D-glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscles and their coenzyme-binding properties

Experimental conditions favouring the dissociation of tetrameric rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase into active monomers were elaborated. The urea-induced dissociation of the tetramer was shown to be a stepwise process (in 2 M urea only dimers are formed; an increase in urea concentration up to 3 M causes the splitting of the dimers into monomers). The specific activity of immobilized monomers in the glyceraldehyde-3-phosphate oxidation reaction does not differ from that of the parent immobilized tetrameric form. The tetrameric enzyme molecule binds the coenzyme with a negative cooperativity (the first two NAD+ molecules bind with KD below 0.1 microM; for the third and fourth molecules the dissociation constant was determined to be equal to 5.5 +/- 1.5 microM (50 mM medinal buffer, 10 mM sodium phosphate, pH 8.2). The cooperativity of NAD+ binding is preserved in the immobilized preparation of tetrameric dehydrogenase. The immobilized monomers bind NAD+ with KD of 1.6 +/- 1.0 microM. The experimental results are consistent with the hypothesis according to which the association of catalytically active subunits into a tetramer changes their coenzyme-binding properties in such a way that the first two NAD+ molecules bind more firmly to a tetramer than to a monomer, whereas the third and the fourth NAD+ molecules bind less firmly.     

Mechanism of palmityl coenzyme A inhibition of liver glycogen synthase.

Palmityl-CoA inhibits free liver glycogen synthase; the concentration required for half-maximum inhibition is 3 to 4 micrometer. Almost complete inhibition was observed at 50 micrometer. Palmityl-CoA inhibition is associated with dissociation of the tetrameric enzyme into monomers, and binding of palmityl-CoA to the monomers. Glycogen-bound enzyme is also inhibited by palmityl-CoA, resulting in dissociation of the enzyme into monomers and concomitant release of the enzyme from the primer glycogen. Palmityl-CoA inhibition of the enzyme is partially reversed by the glycogen synthase activator, glucose-6-P, whereas sodium lauryl sulfate-inhibited enzyme is not reactivated by glucose-6-P. Sodium lauryl sulfate inhibition results in the dissociation of the tetramer into the monomers. Bovine serum albumin and cyclodextrin can prevent palmityl-CoA inhibition only when they are added prior to palmityl-CoA addition. The possible physiological role of palmityl-CoA in glucose homeostasis is discussed.     

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.     

Linked functions in allosteric proteins: Extension of the concerted (MWC) model for ligand-linked subunit assembly and its application to human hemoglobins

The allosteric model of Monod et al. (1965) (MWC) has been extended to take into account the effects of subunit dissociation. The problem is formulated theoretically in terms of a general model for two allosteric species (dimers and tetramers) linked by a polymerization reaction. Relationships are presented for interpreting the dimer-tetramer association constants in terms of allosteric model parameters.Sub-cases of the general model were tested against recent experimental data on the oxygenation-linked dimer-tetramer equilibria in normal human hemoglobin and in the variant hemoglobin Kansas (β₁₀₂, Asp → Thr). The objectives of these analyses were: (1) to find the simplest models capable of describing the linked dimer-tetramer equilibria in the two hemoglobin systems, and (2) to evaluate the corresponding model parameters so that allosteric properties of the two hemoglobins may be compared.In the simplest version of the model, the dimer is half of an R-state tetramer. This model was found to be excluded unequivocally by the data for both normal hemoglobin and hemoglobin Kansas when the α and β chains have equal binding affinities. When this two-state model was modified to permit non-equivalent affinities for the chains, the model could be fitted to hemoglobin Kansas, but not to hemoglobin A. A model, in which the dimers are allowed to exist in a state different from the tetramer R state, was found to be consistent with the data for hemoglobin A, with equivalent binding by the α and β chains. For hemoglobin A, the unliganded R-state tetramers have a different subunit dissociation energy from that of fully liganded R-state tetramers. The simplest model capable of describing both hemoglobin A and hemoglobin Kansas was obtained by extending this three-state model to permit (but not require) functional non-equivalence of the α and β chains. For these MWC models, unique estimates were obtained for the model parameters.The allosteric constants for tetrameric hemoglobins A and Kansas are approximately equal. The value obtained from hemoglobin A is similar to previous estimates, whereas the value for hemoglobin Kansas is lower than previously estimated (Edelstein, 1971) by approximately two orders of magnitude. The low affinity of hemoglobin Kansas tetramer does not arise from an unusually high allosteric constant favoring the T-state species. It is largely the consequence of a greatly reduced oxygen affinity of β chains in the T state, and reduced values for the ratio between affinities in the R and T states.     

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+.     

Destabilization of the Homotetrameric Assembly of 3-Deoxy-d-Arabino-Heptulosonate-7-Phosphate Synthase from the Hyperthermophile Pyrococcus furiosus Enhances Enzymatic Activity

Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), adopts a variety of different quaternary structures depending on the organism in which it is found. The DAH7PS from the hyperthermophilic archaebacterium Pyrococcus furiosus was previously shown to be tetrameric in its crystalline form, and this quaternary association is confirmed in an improved structure in a different crystal system. This tetramer is also present in solution as revealed by small-angle X-ray scattering and analytical ultracentrifugation. This homotetrameric form has two distinct interfaces, both of which bury over 10% each of the surface area of a single monomer. Substitution of Ile for Asp in the hydrophobic region of one interface gives a protein with a remarkable 4-fold higher maximum catalytic rate than the wild-type enzyme. Analytical ultracentrifugation at pH 7.5 reveals that the tetrameric form is destabilized; although the protein crystallizes as a tetramer, equilibrium exists between tetrameric and dimeric forms with a dissociation constant of 22 μM. Thus, under the conditions of kinetic assay, the enzyme is primarily dimeric, revealing that the dimeric form is a fully functional catalyst. However, in comparison to the wild-type protein, the thermal stability of the dimeric protein is significantly compromised. Thus, an unusual compromise of enzymatic activity versus stability is observed for this DAH7PS from an organism that favors a hyperthermophilic environment.     

Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relations to a fetal form

The Novikoff hepatoma glycogen phosphorylase b has been purified over 300-fold, free of glycogen synthetase, some of its properties have been studied, and its relationship to fetal forms of rat muscle and liver phosphorylase has been established immunochemically. Its molecular weight is approximately 200,000, and, like the liver but unlike the muscle isozyme, it does not dimerize on conversion to the a form. However, it differs from the liver isozyme in being activated by AMP (K_a = 0.2 mM) and in not being activated by sulfate ion. Antibody to the adult rat muscle phosphorylase did not inhibit the activity of the tumor or liver isozyme. Although antibody to liver or hepatoma phosphorylase had no effect on adult muscle phosphorylase, each of these antibodies partially inhibited the other enzyme. These findings indicate the presence of some liver isozyme in the tumor, and this was confirmed by isoelectric focusing. Rat liver and muscle phosphorylase (and synthetase) were low during embryonal development but rose rapidly at or shortly after birth. Immunochemical studies revealed that both fetal liver and fetal muscle phosphorylases are immunologically identifiable with the tumor enzyme; and the fetal form is also present as a major form in rat kidney and brain.     

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.