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.

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.

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).

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.

[Delayed activation of muscle glycogen phosphorylase b on incubation of the enzyme with adenosine-5'-monophosphate].

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).

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.

[Mechanism for regulating the activity of muscle glycogen phosphorylase b by adenosine-5'-monophosphate].

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.

[Inhibition of muscle glycogen phosphorylase b by heterocyclic vitamins and coenzymes].

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.

[Inhibition of muscle glycogen phosphorylase b by 5-methyltetrahydrofolic acid, 3'-chloro- and 3',6'-dichloromethotrexates].

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.

[Interaction of muscle glycogen phosphorylase b with riboflavin, its tetraacetyl derivative and their analogs].

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.

[Interaction of muscle glycogen phosphorylase b with nicotinic acid, nicotinamide, N-nicotinyl-gamma-aminobutyric acid and nicotinamide coenzymes].

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.

[Interaction of muscle glycogen phosphorylase B with methotrexate, folic and folinic acids].

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 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].

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.

[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 interaction of muscle glycogen phosphorylase b with glycogen.

Interaction of muscle glycogen phosphorylase b (EC 2.4.1.

[Kinetics of the reaction between muscle glycogen phosphorylase B and glycogen].

Kinetics of glycogen binding by glycogen phosphorylase b has been studied by stopped flow and temperature jump methods. This reaction is followed by increase in light scattering whose amplitude depends upon the enzyme binding sites concentration of glycogen particles occupied by the enzyme. It has been shown that the complex formation has the first order with respect to enzyme and glycogen concentrations.

[Regulation of enzyme activity in adsorptive enzyme systems. II. Influence of dextran sulfate on catalytic properties of lactate dehydrogenase (isozyme M4)].

It has been shown that the binding of pig skeletal muscle lactate dehydrogenase (isozyme M4) by dextran sulfate with weight-average molecular weight 500 000 is accompanied by a decrease of the rate of enzymatic reduction of pyruvate. The hyperbolic dependence of the enzymic reaction rate on NADH concentration observed for free lactate dehydrogenase is transformed in a sigmoidal curve in the case of adsorbed enzyme form (Hill's coefficient is equal to 2.1).