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Based on a theoretical analysis of functioning of a monocascade enzymatic system, a method for continuous monitoring of the phosphorylase kinase-catalyzed enzymatic reaction has been developed. The method is based on the ability of the kinase reaction product--the phosphorylated form of glycogen phosphorylase (form a)-to catalyze glycogen phosphorolysis (with inorganic phosphate as the low molecular weight substrate) or synthesis (with glucose 1-phosphate) in the absence of AMP. A turbidimetric method may be used for the monitoring of the reaction of glycogen degradation (or synthesis) by phosphorylase a formed in the course of the kinase reaction. A method to calculate the initial rate of the kinase reaction from an absorbance versus (time)2 plot has been theoretically substantiated.

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.

The effect of specific ligands (the substrate glucose 1-phosphate, AMP, and flavins) on the initial rate of tryptic digestion of muscle glycogen phosphorylase b has been studied (0.02 M HEPES, pH 6.8; 37 degrees C). Differences were observed between the kinetic curves of the enzyme trypsinolysis initiated by the addition of a trypsin/ligand mixture and once trypsin was added to the enzyme preincubated with a ligand for 10 min. It was suggested that the specific ligands studied induce relatively slow conformational changes in the phosphorylase b molecule.

The two-phase character of essential histidine residues modification of pyruvate dehydrogenase component of pyruvate dehydrogenase complex from pigeon breast muscle by diethylpyrocarbonate has been demonstrated. The relative amplitude of the fast phase increases with increasing the modificator concentration. The model of chemical modification of dimeric enzyme where the modification of the residue in one subunit leads to the change of reactivity of corresponding residue in the other subunit is used for the description of inactivation kinetics. The expression for the diminishing of enzyme activity in the course of chemical modification and the methods of kinetic parameters estimation have been proposed. The following values of kinetic parameters for the modification of pyruvate dehydrogenase component by diethylpyrocarbonate were obtained (pH 6.0; 20 degrees C): k1 = 6400 +/- 400 M-1 min-1 (the microscopic rate constant for the modification of histidine residue in the intact dimer), k2 = 890 +/- 200 M-1 min-1 (the rate constant for the modification of histidine residue in the intact subunit in the dimer which contains one modified subunit) and kt = 0.9 +/- 0.2 min-1 (the rate constant for conformational transition of the dimer induced by modification of histidine residue in one of the subunits in the dimeric molecule).

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.

The notion of the "primary blocks" of cellular metabolism (designated as "metabolic system") has been introduced. Metabolic system is defined as a metabolic pathway which corresponds to the really existing multienzyme complex. The complex of glycolytic enzymes which catalyzes the anaerobic reduction of glucose-6-phosphate with production of ATP may serve as an example of metabolic system (this complex does not contain hexokinase). The complex is formed on thin filaments of I-band of the muscle fibers or on dimers of band 3 protein embedded in the erythrocyte membranes. The fixation of the multienzyme complex to the support of biological nature provides the material basis for regulation of the metabolic system by chemical signals produced by the higher levels of metabolic control. Owing to interaction with anchor protein of the support the chemical signals exert the general control of functioning the multienzyme complex (switching on--switching-off of the metabolic system). It is assumed that the glycolytic system in skeletal muscles is stimulated by Ca2+ ions which interact with the anchor protein of the support (troponin C).

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.

A theoretical analysis has been made of dependencies of specific enzymatic activity (a) on the concentration of the enzyme for associating enzyme systems, in which the association of protein molecules leads to the formation of linear associates of an unlimited length (M in equilibrium M2 in equilibrium M3 in equilibrium ...) and is accompanied by steric shielding of active centers, and also for systems of the type 2 M in equilibrium D in equilibrium D2 in equilibrium D3 in equilibrium ... (M is an inactive monomer and D is an active dimer), in which the specific enzymatic activity of the dimer does not depend on the degree of association. For both models an analysis has been made of the S-shape of the curves of the dependence of a on the concentration of the substrate. Experimental data for glutamate dehydrogenase from ox liver and phosphofructokinase from rabbit skeletal muscles have been used as illustrations.

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.

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.

General properties of enzymes and structurally ordered multienzyme complexes as controllable systems are discussed: the spatial isolation of working sites and sites of control and the realization of control mechanisms with the participation of "external" factors which provide the optimal functioning of the controllable system in the biological system of higher level of complexity. The basic mechanisms of the control of soluble enzymes are isosteric and allosteric mechanisms which directed to the maintenance of cellular homeostasis. The mechanism of functioning of a multienzyme complex as a whole which is realized with the participation of second messengers is classified as a mechanism for tracing of the signals from higher levels of the control of metabolism (from nervous, hormonal and immune systems). When discussing the control of functioning of the multienzyme complexes, special attention was paid to the complex of glycolytic enzymes formed on the structural proteins of skeletal muscles and on the membranes. An order of assembly of the complex of glycolytic enzymes is proposed. The possible localization of this complex in myofibrils is discussed.

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 μmol dimeric glycogen phosphorylase b per g glycogen, which corresponds to 20 dimeric enzyme molecules per average glycogen molecule of M r 5.5 · 10 6 . Microscopic dissociation constants were determined for the enzyme-glycogen complex within the temperature range from 12.7 to 30.0°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°C, whereas the bimolecular rate constant changes sligtly and is about 10 8 M −1 · s −1 . These data point to the possibility of diffusional control of the complex formation.

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. Relaxation kinetics is compatible with proposed bimolecular reaction scheme. Microscopic rate constants of the forward and reverse reactions of glycogen binding by glycogen phosphorylase b are determined in temperature range from 12,7 to 30 degrees C. The possibility of diffusional control of the binding rate is discussed.