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.

Kinetic mechanism of allosteric regulation of muscle glycogen phosphorylase B by adenosine 5'-monophosphate.

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 AMP concentration under the saturation of the enzyme by glycogen. Applicability of some 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 of the enzymatic reaction describing adequately the activation of the enzyme by AMP and inhibition at sufficiently high concentrations of AMP is proposed.

Slow step in activation of muscle glycogen phosphorylase b by adenosine 5'-monophosphate.

The incubation of glycogen phosphorylase b from rabbit skeletal muscles with the allosteric activator (AMP) for 10-15 min has been found to result in an additional increase in the initial rate of the enzymatic reaction (v) as compared with the corresponding value of v measured by the initiation of the enzymatic reaction by the addition of a mixture of glucose 1-phosphate and AMP (0.02 M HEPES, pH 6.8; 37 degrees C). Glycogen with molecular mass of 270.10(6) daltons was used in the kinetic experiments. It is assumed that the enhancement of the rate of the phosphorylase b reaction is due to association of the enzyme molecules adsorbed to a glycogen particle resulting in an increase in the affinity of the enzyme for glycogen.

Specificity of inhibition of muscle glycogen phosphorylase b by flavins.

The inhibition of glycogen phosphorylase b from rabbit skeletal muscles by the derivatives of riboflavin, FMN, FAD, and 2', 3', 4', 5'-tetraacetylriboflavin substituted in positions 6 and 8 of the isoalloxazine part of the flavin molecule is found to be cooperative (the Hill coefficient, h, exceeds 1.0). The modification of the flavin molecule slightly changes the value of the Hill coefficient, but results in the increase of the "half-saturation" concentration [I]0.5.

[Delayed activation of muscle glycogen phosphorylase b on incubation of the enzyme with adenosine-5'-monophosphate]. / Medlennaia aktivatsiia myshechnoi glikogenfosforilazy b pri inkubatsii fermenta s adenozin-5'-monofosfatom.

When studying the enzyme activity of glycogen phosphorylase b from rabbit skeletal muscles by the turbidimetric method or by the method based on the determination of inorganic phosphate (the product of the enzymatic reaction) in the direction of glycogen synthesis we observed that 10-15 min preincubation of the enzyme with the allosteric activator (AMP) results in an increase in the initial rate of the enzymatic reaction (nu) in relation to the corresponding value of nu measured by the initiation of the enzymatic reaction by the addition of the mixture of glucose 1-phosphate and AMP (0.02 M Hepes, pH 6.8; 37 degrees C). Glycogen with molecular mass of (264-276).10(6) dalton was used in the kinetic experiments. For 1 mM AMP the time-dependent process of phosphorylase b activation under the action of AMP follows the exponential law with the apparent rate constant of the first order equal to 0.43 min-1 (turbidimetric method of measurement of enzyme activity). When AMP concentration increases, the degree of activation of phosphorylase b reaches a limiting value equal to 1.75 (6 mM glucose 1-phosphate, 0.2 mg/ml glycogen). The activation effect decreases with increasing glycogen concentration and disappears at saturating concentrations of high-molecular weight substrate. Incubation of phosphorylase b with AMP causes the lowering of Michaelis constant for glucose 1-phosphate. It is assumed that enhancement of the rate of the enzymatic reaction catalyzed by phosphorylase b during incubation with AMP is due to association of the enzyme molecules adsorbed to a glycogen particle resulting in an increase in the affinity of the enzyme for glycogen.

Kinetic mechanism of activation of muscle glycogen phosphorylase b by adenosine 5'-monophosphate.

The kinetic analysis of the enzymatic reaction catalyzed by glycogen phosphorylase b from the rabbit skeletal muscle has been carried out in the direction of glycogen synthesis under the conditions of the saturation of the enzyme by glycogen. The applicability of 12 different variants of the Monod-Wyman-Changeux model and 6 kinetic schemes involving the interaction of AMP- and glucose-1-phosphate-binding sites in the dimeric enzyme molecule is considered. The kinetic model has been proposed, which assumes (i) the independent binding of AMP and one molecule of glucose 1-phosphate with the enzyme saturated by glycogen, (ii) the exclusive binding of the second molecule of glucose 1-phosphate by the enzyme containing two molecules of AMP and one molecule of glucose 1-phosphate, and (iii) the exclusive ability of the complex of the enzyme with glycogen, two molecules of AMP, and two molecules of glucose 1-phosphate to undergo the catalytic transformation. The parameters of the equation of the initial steady-state rate of the enzymatic reaction are calculated by the nonlinear regression method. The proposed kinetic scheme is shown to satisfy to the following criteria: (i) the convergence under the regressional analysis, (ii) the reliability of the values of the parameters of the model, (iii) the minimality of the sum of the weighted squares of the differences between the experimental and calculated values of the reaction rate.

[Mechanism for regulating the activity of muscle glycogen phosphorylase b by adenosine-5'-monophosphate]. / Mekhanizm reguliatsii aktivnosti myshechnoi glikogenfosforilazy b adenozin-5'-monofosfatom.

The kinetics of glycogen chains growth catalyzed by glycogen phosphorylase b from rabbit skeletal muscles at saturating glycogen concentrations have been studied. The applicability of twelve different variants of the Monod-Wyman-Changeux model and six kinetic schemes assuming the interaction of AMP- and glucose 1-phosphate binding sites in the dimeric enzyme molecule is discussed. The kinetic model of the reaction is proposed which suggests: (1) independent binding of AMP and a molecule of glucose 1-phosphate to the dimeric enzyme molecule saturated by glycogen; (2) exclusive binding of the second glucose 1-phosphate molecule by the dimeric enzyme molecule containing two molecules of bound AMP and one molecule of bound glucose 1-phosphate; (3) exclusive ability of the enzyme complex with glycogen, two molecules of AMP and two molecules of glucose 1-phosphate in catalytic transformation. The parameters of the rate equation were calculated by nonlinear regression analysis. The proposed kinetic scheme satisfies the following criteria: (1) convergence in the regressional analysis; (2) reliability of the parameter values; (3) the minimal sum of the weighed squares of residuals. This model was found to fit adequately the kinetics of muscle phosphorylase b reaction at saturating glycogen concentrations.

[Inhibition of muscle glycogen phosphorylase b by heterocyclic vitamins and coenzymes]. / Ingibirovanie myshechnoi glikogenfosfolirazy b geterotsiklicheskimi vitaminami i kofermentmi.

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by biotin, pyridoxine, lipoic acid, as well as by thiamine and cobalamine vitamins and coenzymes has been found. The values of "half-saturation" concentration and Hill coefficients are determined for biotin (27 mM, 1.3), pyridoxine (19 mM, 1.7), 5'-deoxyadenosyl-cobalamine (2.5 mM, 1.5), lipoic acid (3.4 mM, 1.1), thiamine (11 mM, 1.3), thiamine diphosphate (11 mM, 1.0). Effectiveness of the enzyme inhibition by vitamins and coenzymes containing different heterocyclic groups is analysed; riboflavin and its coenzymic forms are suggested to be the most effective inhibitors.

[Inhibition of muscle glycogen phosphorylase b by 5-methyltetrahydrofolic acid, 3'-chloro- and 3',6'-dichloromethotrexates]. / Ongibirovanie myshechnoi glikogenfosforilazy b 5-metiltetragidrofolievoi kislotoi, 3'-khlor- i 3',5'-dikhlormetotreksatami.

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates has been studied. The inhibition is reversible and characterized by positive kinetic cooperativity (Hill coefficient exceeds 1). The values of pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: 3',5'-dichloromethotrexate, 3'-chloromethotrexate, 5-methyl-5,6,7,8-tetrahydrofolic acid (0.24, 0.40 and 1.87 mM, respectively). Comparison of "half-saturation" concentrations for the above compounds and for methotrexate and folinic acid shows that pterin affinity to glycogen phosphorylase b is affected by substituents both in pteridine and in p-aminobenzoic moieties of the pterin molecule. The antagonism between 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates, on the one hand, and AMP and FMN, on the other, is revealed for combined action of modifiers on glycogen phosphorylase b.

[Interaction of muscle glycogen phosphorylase b with riboflavin, its tetraacetyl derivative and their analogs]. / Vzaimodeistvie myshechnoi glikogenfosforilazy b s riboflavinom, ego tetraatsetilproizvodnym i ikhh analogami.

The interaction of rabbit skeletal muscle glycogen phosphorylase b with riboflavin, 2',3',4',5'-tetraacetylriboflavin and their analogues, containing different substituents in the positions 6, 8 and 8 alpha, has been studied. Dissociation constant for the complex of the enzyme and riboflavin was determined to be 12.5 microM (pH 6.8; 20 degrees C) by sedimentation velocity method. Riboflavin and its analogues have been found to inhibit glycogen phosphorylase b. The inhibitor half-saturation concentration values increase in the following order: riboflavin (18 microM), 8-methoxy(nor)rifoblavin (23 microM), 8 alpha-bromo-2',3',4',5'-tetraacetylriboflavin (40 microM), 6-bromoriboflavin (40 microM), 8 alpha-hydroxyriboflavin (60 microM), 8-hydroxy(nor)riboflavin (90 microM), 8 alpha-(gamma-carboxypropylamino-2',3',4',5'-tetraacetylriboflav in (90 microM), 8 alpha-[p-(5-ethyl-1,3,4-thiodiazol-2-ylsulfamido)phenylamino ]- 2',3',4',5'-tetraacetylriboflavin (100 microM), 8 alpha-(L-methionyno)-2',3',4',5'-tetraacetylriboflavin (120 microM), 8 alpha-[p-(thiazol-2-ylsulfamido)phenylamino]- 2',3',4',5'-tetraacetylriboflavin (140 microM), 8 alpha-(p-sulfamidophenylamino)-2',3',4',5'-tetraacetylriboflavi n (180 microM), 8 alpha-(p-carboxyphenylamino)-2',3',4',5'-tetraacetylriboflavin+ ++ (210 microM), 2',3',4',5'-tetraacetylriboflavin (250 microM), 8 alpha-(L-homoserino)-2',3',4',5'-tetraacetylriboflavin (340 microM), 8 alpha-(L-glutamo)-2',3',4',5'-tetraacetylriboflavin (360 microM). The existence of glycogen phosphorylase b complexes with riboflavin and its analogues has been proved by methods of absolute and difference spectrophotometry.

[Interaction of muscle glycogen phosphorylase b with nicotinic acid, nicotinamide, N-nicotinyl-gamma-aminobutyric acid and nicotinamide coenzymes]. / Vzaimodeistvie myshechnoi glikogenfosforilazy b s nikotinovoi kislotoi, nikotinamidom, N-nikotinoil-gamma-aminomaslianoi kislotoi i nikotinamidnymi kofermentami.

The inhibitory action of nicotinic acid, nicotinamide, N-nicotinoyl-gamma-aminobutyric acid, NAD, NADH, NADP, and NADPH on the rabbit skeletal muscle glycogen phosphorylase b has been studied. The inhibition is reversible and positively cooperative (the value of Hill coefficients were determined for the following compounds: nicotinic acid (28 mM; 1.4), nicotinamide (4.4 mM; 1.2), N-nicotinoyl-gamma-aminobutyric acid (9.5 mM; 1.4), NAD (4.4 mM; 1.2), NADH (0.93 mM; 1.2). NADH-binding site of glycogen phosphorylase b subunit was characterized by the sedimentation velocity method. Microscopic dissociation constant was found to be 86 +/- 9 microM (pH 6.8; 20 degrees C). AMP-induced association of glycogen phosphorylase b is hindered by NADH.

[Interaction of muscle glycogen phosphorylase B with methotrexate, folic and folinic acids]. / Vzaimodeistvie myshechnoi glikogenfosforilazy B s metotreksatom, folievoi i folinovoi kislotami.

The interaction of rabbit skeletal muscle glycogen phosphorylase b with methotrexate, folic and folinic acids has been studied. Microscopic dissociation constant for the glycogen phosphorylase b--methotrexate complex determined by analytical ultracentrifugation is 0.43 mM. A subunit of glycogen phosphorylase b is shown to have two sites for methotrexate binding. AMP and FMN diminish the affinity of glycogen phosphorylase b to methotrexate, whereas glycogen does not influence the methotrexate binding to the enzyme. Methotrexate, folic and folinic acids are found to be inhibitors of the muscle glycogen phosphorylase b. The inhibition is reversible and characterized by positive kinetic cooperativity (the Hill coefficient exceeds one unity). The value of the pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: folic acid (0.65 mM), methotrexate (1.01 mM), folinic acid (3.7 mM). The antagonism between methotrexate, folic and folinic acids, on the one hand, and AMP and FMN, on the other, is revealed for their combined action.

A spectrophotometric study of vitamin B2 and its coenzyme forms binding to muscle glycogen phosphorylase b.

The vitamin B2 and its coenzyme forms binding to glycogen phosphorylase b from rabbit skeletal muscle has been studied by the spectrophotometric method. The spectral properties of riboflavin, FMN and FAD bound to muscle glycogen phosphorylase b were found to be identical at the wavelengths of 300 to 500 nm. According to data on spectrophotometric titration of muscle glycogen phosphorylase b by FMN, each subunit of the enzyme contains one flavin-binding site.

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

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

[Interaction of muscle glycogen phosphorylase B with flavin-adenine dinucleotide and its analogs]. / Vzaimodeistvie myshechnoi glikogenfosforilazy B s flavinadenindinukleotidom i ego analogami.

The inhibition of rabbit skeletal muscle glycogen phosphorylase b by FAD and its analogues with substitutes in the position 8 has been studied. The value of half-saturation, [I]0,5, for inhibitors increases in the following order: FAD (44 microM), 8 alpha-hydroxy-FAD (60 microM), 8-dimethylamino (nor)-FAD (69 microM), 8 alpha-(N-acetyl-L-cystein-S-yl)-FAD (106 microM). From the comparison of these values with those obtained earlier for FMN analogues, it follows that in the case of FAD the half-saturation value is less sensitive to modification of the position 8 in the flavin isoalloxazine ring. The existence of the glycogen phosphorylase b FAD-complex has been proved by the spectrophotometry and sedimentation methods. The positions of maxima of optical absorption of the enzyme-bound FAD in the 300-500 nm region are identical with corresponding positions for FMN. FAD has been shown to hinder the AMP-induced transition of dimeric form of the enzyme to tetrameric one.

[Interaction of muscle glycogen phosphorylase B with flavin mononucleotide and its analogs]. / Vzaimodeistvie myshechnoi glikogenfosforilazy B s flavinmononukleotidom i ego analogami.

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.

The interaction of muscle glycogen phosphorylase b with glycogen.

Interaction of muscle glycogen phosphorylase b (EC 2.4.1.1) with glycogen was studied by sedimentation, stopped-flow and temperature-jump methods. The equilibrium enzyme concentration was determined by sedimentation in an analytical ultracentrifuge equipped with absorption optics and a photoelectric scanning system. The maximum adsorption capacity of pig liver glycogen is 3.64 mumol dimeric glycogen phosphorylase b per g glycogen, which corresponds to 20 dimeric enzyme molecules per average glycogen molecule of Mr 5.5 X 10(6). Microscopic dissociation constants were determined for the enzyme-glycogen complex within the temperature range from 12.7 to 30.0 degrees C. Enzyme-glycogen complexing is accompanied by increasing light scattering and its increment depends linearly on the concentration of the binding sites on a glycogen particle that are occupied by the enzyme. Complex formation and relaxation kinetics are in accordance with the proposed bimolecular reaction scheme. The monomolecular dissociation rate constant of the complex increases as the temperature increases from 12.7 to 30.0 degrees C, whereas the bimolecular rate constant changes slightly and is about 10(8) M-1 X S-1. These data point to the possibility of diffusional control of the complex formation.