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The role of multienzyme complexes in integration of cellular metabolism
Z theo~ BioL (1986) 119, 445-455
The Role of Multienzyme Complexes in Integration of Cellular Metabolism B. I. KURGANOV
The All-Union Vitamin Reseach Institute, Moscow, U.S.S.R. (Received 13 July 1985, and in revised form 11 November 1985) The notion of the "primary block" of cellular metabolism designated as "metabolic system" is introduced. Metabolic system is defined as a metabolic pathway which corresponds to the structurally ordered 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/-band of the muscle fibres or on the dimers of band 3 protein embedded in the erythrocyte membrane. The fixation of the multienzyme complex to the support of the 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 ofthe multienzyme complex (switching on-switching off the metabolic system). It is assumed that glycolytic system in skeletal muscles is stimulated by Ca 2÷ ions which interact with the anchor protein of the support (troponin C). 1. Introduction The elucidation of the regulatory mechanisms of cellular metabolism is one of the importan~ problems of modern biological chemistry. At present a great number of experimental data which demonstrate the existence of regulatory relations in the cell has been accumulated. However current knowledge of how each metabolic process is brought to conformity with the needs of the cell and organism is far from complete. In my opinion, the methodological approaches to the study on the mechanisms of regulation of metabolic pathways have not been properly developed. This circumstance impedes the elaboration of the principles of integration of cellular metabolism. The aim of the present paper is to discuss the hierarchy of the levels of organization and control of metabolism. The notion of metabolic system is introduced. Metabolic system is defined as a metabolism fragment which corresponds to a multienzyme complex. The idea is suggested that the general control of functioning metabolic systems is exerted by second messengers.
2. Metabolic Systems The existence of spatial organization of metabolic processes is universally recognized (Siekevitz, 1959; Martin, 1966; Kempner, 1975; Ottaway & Mowbray, 1977, Welch, 1977; Srere, 1985). It is evident that the hierarchy of the levels of organization 445
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and control of metabolism must reflect the hierarchy of the structural levels of matter in the living organisms (enzyme--> multienzyme complex-> subcellular structure--> cell--> tissue--> organ--> functional systems--> • • .) (see, for example, Antonomov, 1977; Konev, 1979). In the present paper the discussion is restricted by the consideration of the level corresponding to the formation of multienzyme complexes. We shall designate metabolic system as a set of metabolic stages catalyzed by the enzymes forming the ordered multienzyme complex. In other words, we must choose metabolic systems in accordance with the data on structural organization of matter in living systems. We shall explain the definition of metabolic system using glycolytic pathway (i.e. the process of anaerobic oxidation of glucose) as an example. Histochemical investigations have indicated that in skeletal muscle the glycolytic enzymes (hexokinase being a prominent exception) are localized within I-band of the muscle fibres (Sigel & Pette, 1969; D/blken et al., 1975). The thin filaments of I-band are composed of F-actin and regulatory proteins (tropomyosin and troponin). The high local concentrations of the glycolytic enzymes in adsorbed state must favour revealing enzyme-enzyme interactions and formation of a structurally ordered multienzyme complex (Pette, 1975). The hypothetical structure of the complex of the glycolytic enzymes was proposed by us earlier (Kurganov, 1984; Kurganov et al., 1985). This complex does not contain hexokinase. It has been assumed that the first step of the formation of the multienzyme complex is the interaction of 6-phosphofructokinase with the anchor protein of the support. The tetrameric molecule of 6-phosphofructokinase possesses three mutually perpendicular symmetry axes of second order. The adsorption of 6-phosphofructokinase takes place along one of the symmetry axes of the enzyme molecule and is followed by the binding of glucosephosphate isomerase, pyruvate kinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, lactate dehydrogenase, enolase, triosephosphate isomerase and glycerol-3-phosphate dehydrogenase (two molecules of each enzyme per multienzyme complex). The complex of glycolytic enzymes has symmetry axis of second order. The formation of the multienzyme complex results in the creating a microcompartment where glycolysis proceeds without release of glycolytic intermediates into the medium. Thus, if one keeps the suggested definition of "metabolic system", glycolytic pathway, strictly speaking, is not a metabolic system. In this case a metabolic system is a system which corresponds to the complex of glycolytic enzymes catalyzing the conversion of glucose-6-phosphate into fructose-6-phosphate and the following stages of glycolysis. The first enzyme of the glycolytic pathway, hexokinase, is probably a part of other metabolic systems.
3. Regulation of Metabolic Systems The mechanisms of regulation of cellular metabolism based on the control of the rates of metabolic processes by cellular metabolites have been studied in detail (Newsholme & Start, 1973; Kurganov, 1982). In multicellular organisms there are the higher levels of regulatory mechanisms which are realized with the participation
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of the signal metabolites (histohormones, neurotransmitters, hormones, hormonoids and antibodies). The signal metabolites ("first messengers") produced by nervous, and immune endocrine systems affect the cell receptor apparatus which transforms the external signals into the language of "own" cellular metabolism. The main intracellular mediators of action of signal metabolites (i.e. "second messengers") are calcium ions which penetrate into the cell through the special channels in the membrane, cyclic AMP produced by the adenylate cyclase system (Iyengar et al., 1980), inositol-l,4,5-triphosphate and diacylglycerol whose appearance is a result of hydrolysis of phosphoinositides (Berridge, 1983, 1984; Joseph, 1984). If one defines a metabolic system as a metabolism fragment which corresponds to a certain level of the structural organization of the matter, it is reasonable to assume that the metabolic system must be regulated as a whole by the signals produced by the higher levels of the control of metabolism. The material basis of the realization of such a regulatory mechanism is the fixation of multienzyme complexes on the definite supports. In my opinion, the physiological sense of the formation of the complex of the enzymes participating in a common metabolic pathway on the support of the biological nature is that the cell receives the possibility of regulating the metabolic system as a whole with the aid of chemical signals (or other material factors) affecting the anchor protein of the support. Consider the ways of the regulation of glycolysis (more strictly, the pathway of the conversion of glucose-6-phosphate to lactate) as a whole in the skeletal muscles. The concentration of C a 2+ ions in the resting muscle is about 10-7 M. The increase in the content of C a 2+ ions up to 10-5 M in response to nervous excitation initiates the muscle contraction which is realized with the use of the energy of ATP hydrolysis. The activation of myosin ATPase by actin becomes possible after the binding of C a 2÷ ions by troponin C. It is evident that C a 2+ ions must stimulate glycolysis,? one of whose products is ATP. The possibility of such a stimulation of glycolysis is understandable if one takes into account that the complex of glycolytic enzymes is formed on the thin filaments of I-band of muscle fibres, one of whose components is troponin C. The latter belongs to anchor proteins ensuring the fixation of the complex of glycolytic enzymes on the support. Therefore one can assume that the glycolytic complex remains inactive, until the binding of C a 2+ ions by the anchor protein of the support (troponin C) takes place. ATP produced in the microcompartment of the complex of the glycolytic enzymes may be transferred directly to ATPase active site of myosin molecule. It is of interest that according to Walsh et al., (1977) fructose-bisphosphate aldolase becomes sensitive to C a 2+ ions after the adsorption to filaments formed by F-actin, troponin and tropomyosin ( C a 2+ ions increase the affinity of adsorbed enzyme to substrate, fructose-l,6-bisphosphate). One can regard these experimental data as a support of the suggestion that glycolysis in skeletal muscles becomes sensitive to C a 2+ owing to the fixation of the complex of glycolytic enzymes on the structural proteins of the muscle. t We do not discuss here the known way of the stimulation of the involvement of glucose residues in glycolytic pathway through glycogen-phosphorylase and phosphoglucomutase reactions by Ca 2+ ions (this way is realized due to the activation of glycogen phosphorylase kinase by Ca 2+ ions).
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If the Ca 2+ ion is the chemical signal which stimulates glycolysis in skeletal muscle as a whole, a signal with the opposite character of action must exist. The effective control may be achieved only on the basis of action of the signals causing the opposite effects. It is known that troponin I and troponin T may be phosphorylated in skeletal and cardiac muscles under the action of cyclic AMP-dependent protein kinase (Adelstein & Eisenberg, 1980). The question of the biological significance of the phosphorylation of troponins I and T in skeletal muscles remains open. An increase in cardiac tropononin I phosphorylation results in a decrease in the actin-activated ATPase activity at a given Ca 2÷ concentration compared to that found with unphosphorylated troponin I (Adelstein & Eisenberg, 1980). It should be noted that cyclic AMP-dependent phosphorylation of 6-phosphofructokinase (this enzyme, in our view, occupies in the complex the position nearest to the anchor protein) is accompanied by the decrease of catalytic efficiency of the enzyme revealing in increase in the sensitivity to inhibition by the excess of ATP and the diminishing the affinity to fructose-6-phosphate (Kitajima et al., 1983). On the basis of these results one can suppose that cyclic AMP activating the cyclic AMPdependent protein kinases is a factor which suppresses the glycolytic process as a whole in skeletal muscles. The investigations of the influence of Ca 2+ on catalytic properties of 6-phosphofrutokinase adsorbed to F-actin-troponin-tropomyosin complex (with use of 6phosphofructokinase and troponins I and T in phosphorylated and dephosphorylated forms) may be valuable for checking the proposed role of Ca 2÷ ions and cyclic AMP in the regulation of glycolysis in skeletal muscles. It should be noted that Ca 2+ ions in concentrations up to 10-5 M do not affect the enzymic activity of free muscle 6-phosphofructokinase (Vaughan et al., 1973).
4. The Role of Aliosteric and Isosteric Regulatory Mechanisms in Functioning Multienzyme Complexes It is known that activities of glycolytic enzymes are regulated by glycolytic intermediates in accordance with isosteric and allosteric regulatory mechanisms which are realized owing to binding of specific ligands in active or allosteric sites of enzyme molecules. All glycolytic enzymes forming the multienzyme complex possess the subunit structure (the only exception is phosphoglycerate kinase). This circumstance results in that one part of active and allosteric sites of the enzymes faces internal microcompartment of the multienzyme complex, whereas the other part appears on the surface of the complex. Perhaps there is no accumulation of intermediates in the microcompartment because the intermediates move as on the conveyor to active sites of the enzymes which catalyze their subsequent conversion. Therefore one can assume that metabolite-regulators realize their action through the sites located on the surface of the multienzyme complex. Such regulatory mechanisms provide the sensitivity of functioning of the multienzyme complex to the metabolite-regulators in the surroundings of the complex. In accordance with above considerations one can distinguish two levels of the control of functioning of the multienzyme complex fixed on the support. The rough
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regulation (switching on-switching off the metabolic system) is exerted by chemical signals affecting the anchor protein of the support. The regulation by metabolites which interact with binding sites located on the surface on the complex ensures the fine adjustment of functioning of the multienzyme complex. These types of regulation differ also in that the power of chemical signals varies in sufficientlywide limits in the cell whereas the concentrations of the key metaboliteregulators (for example, the cofactors of enzymic reactions) are maintained at the constant levels at least during relatively short time intervals (Newsholme & Start, 1973). This circumstance raises certain difficulties for investigators who try to construct the theory of the control of a metabolic system on the basis of regulation of the key enzyme of the metabolic system by metabolic intermediates. It has been known for many years that increased mechanical activity does not lead to large decreases in the content of ATP in the muscle tissue despite the very large increase in energy expenditure. It is exceedingly unlikely for usual mechanisms of interaction of allosteric effectors with enzymes that the small changes in ATP content observed in the experiments could explain the large increases in activity of 6-phosphofructokinase (the key enzyme of glycolysis). Newsholme & Start (1973) have proposed, for example, the mechanism of the amplification of the control of 6-phosphofructokinase activity under the action of ATP. The special role in the realization of this mechanism is assigned to AMP which acts as an activator in the region of the inhibitory concentrations of ATP. However it should be noted that any amplification of the sensitivity of 6-phosphofructokinase reaction to variation of ATP content must result in the enhancement of the contribution of this reaction in the stabilization of the ATP level in the cell. The above difficulties may be avoided if one assumes that the stimulation of the glycolytic process generating ATP for muscle contraction is exerted by Ca 2+ ions affecting the anchor protein which ensures the fixation of the multienzyme complex on the support.
5. The Peculiarities of Functioning Multienzyme Complexes One can expect that the multienzyme complex fixed on the biological support will function in close cooperation with the support. The interactions between the components of the complex must provide the coordination in catalytic action of the enzymes. It is evident that the usual approaches elaborated for analysis of kinetic behaviour of the systems with homogeneous distribution of the enzymes cannot be used for description of functioning of the multienzyme complex fixed on the support. The new approaches must be applied with this aim. One of the approaches may be based on the consideration of the enzyme complex states which differ in the degree of the completeness of catalytic process for individual enzymes of the complex. The interaction of the initial state of the complex with the first substrate of the metabolic pathway is bimolecular reaction. The following transformations of the complex may be considered as the monomolecular transitions which are accompanied by the transfer of the intermediates from one enzyme to the active site of the neighbouring enzyme. Such an approach was used, for example, for description of the electron
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transfer between structurally-bound carriers of the mitochondrial respiratory chain (Rubin & Shinkarev, 1984). Another approach which may be useful, in our opinion, for description of functioning of the multienzyme complex fixed on the support as a whole is based on the application of theory of autowaves (Zhabotinskii, 1974; Ivanitskii et al., 1978). The multienzyme complex may be considered as an excitable medium, each of whose elements (i.e. enzymes) is able to exist in one of three qualitatively different states: the state of rest (the enzyme contains the bound substrate and is ready to perform the catalytic act); the state of excitation (the performing of the catalytic act); and the state of refractoriness (the removal of the products of the reaction from the active site). The attachment of the substrate to the enzyme means the transition from the state of refractoriness into the state of rest. The wave of excitation in the multienzyme complex is initiated by binding of a corresponding chemical signal to the anchor protein of the support to which the multienzyme complex is attached. The passing of the excitation wave in the multienzyme complex initiates the metabolic process in the microcompartment formed by this complex. It should be noted that the properties of the autowave (the rate, the form of the profile, the amplitude) do not depend on the initial conditions initiating it and are determined by the features of the medium, i.e. by the catalytic characteristics of the enzymes, by the character of the interactions of the enzymes in the complex and by the influence of the metabolite-regulators bound to the sites located on the surface of the complex. The initial function of the enzymes in the multienzyme complex takes place in the region of excitation of the autowave. The order of functioning of the enzymes is determined by the structure of the multienzyme complex and the character of moving the autowave and may differ from the order of the positions of the enzymes in the metabolic pathway. The enzyme which directly interacts with the anchor protein of the support (i.e. 6-phosphofructokinase in the complex of glycolytic enzymes) is initiated in the first instance. The changes in the conformation of 6-phosphofructokinase molecule accompanying the catalytic act provoke the transition of neighbour enzymes (glucosephosphate isomerase, pyruvate kinase, fructose-bisphosphate aldolase, lactate dehydrogenase) in the state of excitation. Further extension of the autowave is exerted as a result of conformational changes of the enzymes proceeding to catalytic conversion of the substrates. The transfer of the intermediates on the conveyor takes place probably in the state of the refractoriness. The duration of the refractoriness state is longest for 6-phosphofructokinase which is characterized by the lowest turnover number among the glycolytic enzymes. One can assume that the passing of the excitation wave in the glycolytic enzymes complex fixed on structural proteins of skeletal muscle is coupled with sliding of actin and myosin filaments past each other. 6. The Role of Nature of the Support
It should be noted that the same multienzyme complex may be fixed on different supports. For example, the complex of glycolytic enzymes is formed on the thin filaments of I-band of muscle fibres and on the dimers of band 3 protein (the
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anion-transport system) embedded in the e~throcyte membrane (Green et al., 1965; Tillman et al., 1975; Kurganov, 1984; Kurganov et al., 1985). The attachment of the complex of glycolytic enzymes to the dimers of band 3 protein is shown in Fig. 1.
~
--
l lllllll II II IIIIIIl l l t t l RWgll gll !~, "~
l l l l Illll~U IlI~/WMNNIIJ ml
inside
II ~f IllntlllllllRIiiIlnllmulgllgR
UU u l s m ~ w m m ~ l t outside
FIG. 1. The schematic representation of the attachment of the complex of glycol~ic enzymes to the dimers of band 3 protein embedded in the erythrocyte membrane. Designations: 1 is band 3 protein, 2 is glucosephosphate isomerase, 3 is 6-phosphofructokinase, 4 is fructose-bisphosphate aldolase, 5 is glyceraldehyde-phosphate dehydrogenase, 6 is phosphoglycerate kinase, 7 is phosphoglyceromutase, 8 is enolase, 9 is pyruvate kinase, 10 is lactate dehydrogenase, 11 is triosephosphate isomerase and 12 is glycerol-3-phosphate dehydrogenase.
The nature of second messengers is the same in cells of different types. So the anchor proteins in different matrices are controlled by the same chemical signals. It is known that the band 3 protein is able to bind Ca 2÷ ions which have the inhibitor), effect on the transport of anions through the erythrocyte membrane (Passing & Schubert, 1983). One can expect that the functioning of the glycolytic complex fixed on dimers of band 3 protein will be sensitive to Ca 2÷ ions as in the case of the complex adsorbed to the thin filaments of the muscle fibres. It seems ve~ likely that the functioning of the multienzyme complexes fixed on the anchor proteins of different nature will have specific peculiarities. If the integral membrane-bound proteins play the role of the anchor sites, the formation of the multienzyme complex on the support and its functioning will be influenced by the composition, fluidity and phase state of lipid components of the membrane. The oligomeric state of the anchor protein, its ability to interact with the peripheral enzymes and the chemical signals depend on its microenvironment in the membrane. Many integral membrane-bound enzymes function as systems exerting the transport of certain metabolites through the membrane. The adsorption of the peripheral enzymes to membrane-bound protein channels results in the formation of a united "transport-metabolic system" which provides the functional linkage of metabolic processes proceeding in the cell compartments separated by the membrane. The channel-forming protein plays the role of the "control centre" of a similar system.
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B.I. KURGANOV 7. The Interactions of Metabolic Systems
When explaining the interactions of the metabolic systems one usually takes into account the regulatory relationships which are exerted with the participation of the common metabolites. In my opinion, such an approach is simplified. It is impossible to understand the interrelationships between the metabolic systems without eliciting the chemical signals regulating each of the metabolic systems as a whole. Consider, for example, two metabolic systems (M~ and M2) ensuring the energetic requirements of the cell. The coordinated functioning of both metabolic systems which provides the effective using of the degradable substrates and the stabilization of the levels of the metabolite-indicators of the cell energetic state may be exerted by influence of the chemical signals produced by the higher levels of the control of metabolism upon the anchor proteins of the supports. The nature of these anchor proteins (f/~ and 1/2 in Fig. 2) and the character of their interactions with the chemical signals (X~ and X2) are different. In order to switch on one of the metabolic system with simultaneous switching off of the other metabolic system the chemical signals must exist which are characterized by the opposite character of action on these metabolic systems. Each of the metabolic systems represented schematically in Fig. 2 has two types of signals one of which stimulates and the other suppresses the rate of a given metabolic pathway.
FIG.2. The schemewhichillustratesthe coordinatedfunctioningof two metabolicsystems(MI and M2) fixedto the anchor proteins of the support (f~ and f~2)-X~ and X~ represent the chemicalsignals whichregulatethe metabolicsystemsby the actionon the anchorproteinsof the supports (+ = activation, - = inhibition). It should be noted that the regulatory relations must exist which coordinate the development of the signals of the opposite types. It is known, for example, that the phosphorylation of membrane proteins catalyzed by cyclic AMP-dependent protein kinases causes the change in the permeability of the membrane with respect to Ca 2+ ions. On the other hand, Ca 2+ ions affect the activity of adenylate cyclase. Ca 2+ ions in relatively high concentration inhibit adenylate cyclase, but low concentrations of Ca 2+ ions have the activating effect. The second effect is mediated by calmodulin. The complex of Ca 2+ with calmodulin activates cyclic AMP phosphodiesterase which destroys cyclic AMP (Berridge, 1975; Rodbell, 1983; Tkachuk, 1983). One must take into account the feedbacks between a metabolic system (the object of the control) and the systems transforming the signals from the higher levels of
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the control of metabolism. The stimulation of a metabolic system by chemical signals interacting with the anchor protein of the support must inevitably cause the intensification of the processes directed to decreasing the power of incoming chemical signal. The stimulation of glycolytic process results in the enhancement of production of ATP which may be used by Ca2+-ATPase for pumping off Ca 2+ from sarcoplasm into the contents of the vesicles of sarcoplasmic net. One of the important problems of current metabolism is the underestanding of the control of futile cycle elements. As an example we can mention the cyclic interconversions between fructose-6-phosphate and fructose-l,6-bisphosphate. The phosphorylatio n of fructose-6-phosphate into fructose-l,6-bisphosphate is catalyzed by 6-phosphofructokinase (the enzyme of the glycofytic pathway). The hydrolysis of fructose-l,6-bisphosphate to fructose 6-phosphate is catalyzed by fructose-l,6bisphosphatase (the enzyme of gluconeogenic pathway). The cyclic interconversions between these substrates are accompanied by useless expenditure of ATP and therefore this cycle must be strictly controlled. On the basis of the fact of the complexation between 6-phosphofructokinase and fructose-l,6-bisphosphatase (Uyeda & Luby, 1974; Proffitt et al., 1976) one can propose the existence of the united complex of glycolytic and gluconeogenic enzymes. The effective regulation of the interconversions of fructose-6-phosphate and fructose-l,6-bisphosphate may be achieved with the participation of the anchor protein to which the complex is attached. It seems very likely that the coordinated proceeding the glycolytic and gluconeogenic pathways depends on the state of the anchor protein interacting with second messengers. In summary, we suppose that the rough coordination in functioning of the metabolic systems is exerted with the participation of the signals aitecting the "control centres" of these systems, the anchor proteins of the support (this is the "high" level of the control). The regulatory relations which are realized with the participation of the common metabolite-regulators ensure the fine coordination in functioning of the metabolic systems (the "low" level of the control). These levels of the control are characterized by difterent rates of response. In accordance with the modern data of the temporary organization of the living systems the time constant for each high level of the control is much greater than the analogous value for the lower level (Gadzhiev & Chernyshev, 1976).
8. Conclusion
The principles of the control of metabolic systems proposed in the present paper may be useful for the formulation of the general laws of integration of cellular metabolism. The progress in this field will be determined by obtaining the information about the presence of multienzyme complexes in the cell Only on the basis of similar information can one objectively select the "primary blocks" in cellular metabolism--the metabolic systems. The elucidation of the nature of the support which fixes the multienzyme complex gives the key to the understanding of the mechanisms which exert the switching on or switching off the metabolic system.
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I n m o d e r n b i o l o g i c a l c h e m i s t r y there is n o t a b r a n c h w h i c h d e a l s with the c h e m i c a l signals ( a n d o t h e r m a t e r i a l factors) w h i c h c o n t r o l certain m e t a b o l i c system as a whole. T h e m a j o r i t y o f such c o m p o u n d s ( C a 2÷ ions, cyclic A M P a n d others) are k n o w n b u t we d o n o t y e t realize their role as signals w h i c h switch o n o r switch off m e t a b o l i c systems. T h e e l a b o r a t i o n o f this b r a n c h o f b i o l o g i c a l c h e m i s t r y will m e a n the c o n s i d e r a b l e a d v a n c e in the u n d e r s t a n d i n g o f the m e c h a n i s m s o f the i n t e g r a t i o n of cellular metabolism. When constructing models of regulation of cellular metabolism, one must pay special a t t e n t i o n to the p r o b l e m o f s p a t i a l o r g a n i z a t i o n o f the m e t a b o l i c systems u n d e r study. The m o d e l s with h o m o g e n e o u s d i s t r i b u t i o n o f the e n z y m e s have l i m i t e d value. T h e t h e o r y o f i n t e g r a t i o n o f c e l l u l a r m e t a b o l i s m m u s t be e l a b o r a t e d on the basis o f t h e o r y o f m u l t i l e v e l h i e r a r c h i c a l systems a n d the t h e o r y o f the c o n t r o l h i e r a r c h y in s u c h systems ( W a t e r m a n , 1968; M e s a r o v i r , 1968; G a d z h i e v & C h e r n y shev, 1976; K o n e v , 1979).
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SIEKEVITZ, P. (1959). In: Ciba Foundation Symposium on the Regulation of Cell Metabolism. (Wolstenholme, G. W. W. & O'Connor, C. M, eds). p. 17. London: J. & A. Churchill. SRERE, P. (1985). In: Organized Multienzyme Systems: Catalytic Properties. (Welch, G. R. ed.). p. 1. New York: Academic Press. TILLMAN, W., CORDUA, A. & SCHRt~TER, W. (1975). Biochim. biophys. Acta 383, 157. TKACHUK, V. A. (1983). Introduction in Molecular Endocrinology. (In Russian). Izd. Moscow University. UYEDA, K. & LUBY, L. J. (1974). J. biol. Chem. 249, 4562. VAUGHAN, H., THORNTON. S. D. & NEWSHOLME, E. A. (1973). Biochem. J. 132, 527. WALSH, T. P., CLARKE, F. M. & MASTERS, C. J. (1977). Biochem. J. 165, 165. WATERMAN, T. H. (1968). In: Systems Theory and Biology. p. 1. (Mesarovi~, M. D.ed.). p. 1. Berlin: Spdnger-Verlag. WELCH, G. R. (1977). Prog. Biophys. mol. Biol. 32, 103. ZHABOTINSKII, A. M. (1974). Concentration Autooscillations. (In Russian). Moscow: Nauka.
The Role of Multienzyme Complexes in Integration of Cellular Metabolism B. I. KURGANOV
The All-Union Vitamin Reseach Institute, Moscow, U.S.S.R. (Received 13 July 1985, and in revised form 11 November 1985) The notion of the "primary block" of cellular metabolism designated as "metabolic system" is introduced. Metabolic system is defined as a metabolic pathway which corresponds to the structurally ordered 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/-band of the muscle fibres or on the dimers of band 3 protein embedded in the erythrocyte membrane. The fixation of the multienzyme complex to the support of the 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 ofthe multienzyme complex (switching on-switching off the metabolic system). It is assumed that glycolytic system in skeletal muscles is stimulated by Ca 2÷ ions which interact with the anchor protein of the support (troponin C). 1. Introduction The elucidation of the regulatory mechanisms of cellular metabolism is one of the importan~ problems of modern biological chemistry. At present a great number of experimental data which demonstrate the existence of regulatory relations in the cell has been accumulated. However current knowledge of how each metabolic process is brought to conformity with the needs of the cell and organism is far from complete. In my opinion, the methodological approaches to the study on the mechanisms of regulation of metabolic pathways have not been properly developed. This circumstance impedes the elaboration of the principles of integration of cellular metabolism. The aim of the present paper is to discuss the hierarchy of the levels of organization and control of metabolism. The notion of metabolic system is introduced. Metabolic system is defined as a metabolism fragment which corresponds to a multienzyme complex. The idea is suggested that the general control of functioning metabolic systems is exerted by second messengers.
2. Metabolic Systems The existence of spatial organization of metabolic processes is universally recognized (Siekevitz, 1959; Martin, 1966; Kempner, 1975; Ottaway & Mowbray, 1977, Welch, 1977; Srere, 1985). It is evident that the hierarchy of the levels of organization 445
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and control of metabolism must reflect the hierarchy of the structural levels of matter in the living organisms (enzyme--> multienzyme complex-> subcellular structure--> cell--> tissue--> organ--> functional systems--> • • .) (see, for example, Antonomov, 1977; Konev, 1979). In the present paper the discussion is restricted by the consideration of the level corresponding to the formation of multienzyme complexes. We shall designate metabolic system as a set of metabolic stages catalyzed by the enzymes forming the ordered multienzyme complex. In other words, we must choose metabolic systems in accordance with the data on structural organization of matter in living systems. We shall explain the definition of metabolic system using glycolytic pathway (i.e. the process of anaerobic oxidation of glucose) as an example. Histochemical investigations have indicated that in skeletal muscle the glycolytic enzymes (hexokinase being a prominent exception) are localized within I-band of the muscle fibres (Sigel & Pette, 1969; D/blken et al., 1975). The thin filaments of I-band are composed of F-actin and regulatory proteins (tropomyosin and troponin). The high local concentrations of the glycolytic enzymes in adsorbed state must favour revealing enzyme-enzyme interactions and formation of a structurally ordered multienzyme complex (Pette, 1975). The hypothetical structure of the complex of the glycolytic enzymes was proposed by us earlier (Kurganov, 1984; Kurganov et al., 1985). This complex does not contain hexokinase. It has been assumed that the first step of the formation of the multienzyme complex is the interaction of 6-phosphofructokinase with the anchor protein of the support. The tetrameric molecule of 6-phosphofructokinase possesses three mutually perpendicular symmetry axes of second order. The adsorption of 6-phosphofructokinase takes place along one of the symmetry axes of the enzyme molecule and is followed by the binding of glucosephosphate isomerase, pyruvate kinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, lactate dehydrogenase, enolase, triosephosphate isomerase and glycerol-3-phosphate dehydrogenase (two molecules of each enzyme per multienzyme complex). The complex of glycolytic enzymes has symmetry axis of second order. The formation of the multienzyme complex results in the creating a microcompartment where glycolysis proceeds without release of glycolytic intermediates into the medium. Thus, if one keeps the suggested definition of "metabolic system", glycolytic pathway, strictly speaking, is not a metabolic system. In this case a metabolic system is a system which corresponds to the complex of glycolytic enzymes catalyzing the conversion of glucose-6-phosphate into fructose-6-phosphate and the following stages of glycolysis. The first enzyme of the glycolytic pathway, hexokinase, is probably a part of other metabolic systems.
3. Regulation of Metabolic Systems The mechanisms of regulation of cellular metabolism based on the control of the rates of metabolic processes by cellular metabolites have been studied in detail (Newsholme & Start, 1973; Kurganov, 1982). In multicellular organisms there are the higher levels of regulatory mechanisms which are realized with the participation
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of the signal metabolites (histohormones, neurotransmitters, hormones, hormonoids and antibodies). The signal metabolites ("first messengers") produced by nervous, and immune endocrine systems affect the cell receptor apparatus which transforms the external signals into the language of "own" cellular metabolism. The main intracellular mediators of action of signal metabolites (i.e. "second messengers") are calcium ions which penetrate into the cell through the special channels in the membrane, cyclic AMP produced by the adenylate cyclase system (Iyengar et al., 1980), inositol-l,4,5-triphosphate and diacylglycerol whose appearance is a result of hydrolysis of phosphoinositides (Berridge, 1983, 1984; Joseph, 1984). If one defines a metabolic system as a metabolism fragment which corresponds to a certain level of the structural organization of the matter, it is reasonable to assume that the metabolic system must be regulated as a whole by the signals produced by the higher levels of the control of metabolism. The material basis of the realization of such a regulatory mechanism is the fixation of multienzyme complexes on the definite supports. In my opinion, the physiological sense of the formation of the complex of the enzymes participating in a common metabolic pathway on the support of the biological nature is that the cell receives the possibility of regulating the metabolic system as a whole with the aid of chemical signals (or other material factors) affecting the anchor protein of the support. Consider the ways of the regulation of glycolysis (more strictly, the pathway of the conversion of glucose-6-phosphate to lactate) as a whole in the skeletal muscles. The concentration of C a 2+ ions in the resting muscle is about 10-7 M. The increase in the content of C a 2+ ions up to 10-5 M in response to nervous excitation initiates the muscle contraction which is realized with the use of the energy of ATP hydrolysis. The activation of myosin ATPase by actin becomes possible after the binding of C a 2÷ ions by troponin C. It is evident that C a 2+ ions must stimulate glycolysis,? one of whose products is ATP. The possibility of such a stimulation of glycolysis is understandable if one takes into account that the complex of glycolytic enzymes is formed on the thin filaments of I-band of muscle fibres, one of whose components is troponin C. The latter belongs to anchor proteins ensuring the fixation of the complex of glycolytic enzymes on the support. Therefore one can assume that the glycolytic complex remains inactive, until the binding of C a 2+ ions by the anchor protein of the support (troponin C) takes place. ATP produced in the microcompartment of the complex of the glycolytic enzymes may be transferred directly to ATPase active site of myosin molecule. It is of interest that according to Walsh et al., (1977) fructose-bisphosphate aldolase becomes sensitive to C a 2+ ions after the adsorption to filaments formed by F-actin, troponin and tropomyosin ( C a 2+ ions increase the affinity of adsorbed enzyme to substrate, fructose-l,6-bisphosphate). One can regard these experimental data as a support of the suggestion that glycolysis in skeletal muscles becomes sensitive to C a 2+ owing to the fixation of the complex of glycolytic enzymes on the structural proteins of the muscle. t We do not discuss here the known way of the stimulation of the involvement of glucose residues in glycolytic pathway through glycogen-phosphorylase and phosphoglucomutase reactions by Ca 2+ ions (this way is realized due to the activation of glycogen phosphorylase kinase by Ca 2+ ions).
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If the Ca 2+ ion is the chemical signal which stimulates glycolysis in skeletal muscle as a whole, a signal with the opposite character of action must exist. The effective control may be achieved only on the basis of action of the signals causing the opposite effects. It is known that troponin I and troponin T may be phosphorylated in skeletal and cardiac muscles under the action of cyclic AMP-dependent protein kinase (Adelstein & Eisenberg, 1980). The question of the biological significance of the phosphorylation of troponins I and T in skeletal muscles remains open. An increase in cardiac tropononin I phosphorylation results in a decrease in the actin-activated ATPase activity at a given Ca 2÷ concentration compared to that found with unphosphorylated troponin I (Adelstein & Eisenberg, 1980). It should be noted that cyclic AMP-dependent phosphorylation of 6-phosphofructokinase (this enzyme, in our view, occupies in the complex the position nearest to the anchor protein) is accompanied by the decrease of catalytic efficiency of the enzyme revealing in increase in the sensitivity to inhibition by the excess of ATP and the diminishing the affinity to fructose-6-phosphate (Kitajima et al., 1983). On the basis of these results one can suppose that cyclic AMP activating the cyclic AMPdependent protein kinases is a factor which suppresses the glycolytic process as a whole in skeletal muscles. The investigations of the influence of Ca 2+ on catalytic properties of 6-phosphofrutokinase adsorbed to F-actin-troponin-tropomyosin complex (with use of 6phosphofructokinase and troponins I and T in phosphorylated and dephosphorylated forms) may be valuable for checking the proposed role of Ca 2÷ ions and cyclic AMP in the regulation of glycolysis in skeletal muscles. It should be noted that Ca 2+ ions in concentrations up to 10-5 M do not affect the enzymic activity of free muscle 6-phosphofructokinase (Vaughan et al., 1973).
4. The Role of Aliosteric and Isosteric Regulatory Mechanisms in Functioning Multienzyme Complexes It is known that activities of glycolytic enzymes are regulated by glycolytic intermediates in accordance with isosteric and allosteric regulatory mechanisms which are realized owing to binding of specific ligands in active or allosteric sites of enzyme molecules. All glycolytic enzymes forming the multienzyme complex possess the subunit structure (the only exception is phosphoglycerate kinase). This circumstance results in that one part of active and allosteric sites of the enzymes faces internal microcompartment of the multienzyme complex, whereas the other part appears on the surface of the complex. Perhaps there is no accumulation of intermediates in the microcompartment because the intermediates move as on the conveyor to active sites of the enzymes which catalyze their subsequent conversion. Therefore one can assume that metabolite-regulators realize their action through the sites located on the surface of the multienzyme complex. Such regulatory mechanisms provide the sensitivity of functioning of the multienzyme complex to the metabolite-regulators in the surroundings of the complex. In accordance with above considerations one can distinguish two levels of the control of functioning of the multienzyme complex fixed on the support. The rough
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regulation (switching on-switching off the metabolic system) is exerted by chemical signals affecting the anchor protein of the support. The regulation by metabolites which interact with binding sites located on the surface on the complex ensures the fine adjustment of functioning of the multienzyme complex. These types of regulation differ also in that the power of chemical signals varies in sufficientlywide limits in the cell whereas the concentrations of the key metaboliteregulators (for example, the cofactors of enzymic reactions) are maintained at the constant levels at least during relatively short time intervals (Newsholme & Start, 1973). This circumstance raises certain difficulties for investigators who try to construct the theory of the control of a metabolic system on the basis of regulation of the key enzyme of the metabolic system by metabolic intermediates. It has been known for many years that increased mechanical activity does not lead to large decreases in the content of ATP in the muscle tissue despite the very large increase in energy expenditure. It is exceedingly unlikely for usual mechanisms of interaction of allosteric effectors with enzymes that the small changes in ATP content observed in the experiments could explain the large increases in activity of 6-phosphofructokinase (the key enzyme of glycolysis). Newsholme & Start (1973) have proposed, for example, the mechanism of the amplification of the control of 6-phosphofructokinase activity under the action of ATP. The special role in the realization of this mechanism is assigned to AMP which acts as an activator in the region of the inhibitory concentrations of ATP. However it should be noted that any amplification of the sensitivity of 6-phosphofructokinase reaction to variation of ATP content must result in the enhancement of the contribution of this reaction in the stabilization of the ATP level in the cell. The above difficulties may be avoided if one assumes that the stimulation of the glycolytic process generating ATP for muscle contraction is exerted by Ca 2+ ions affecting the anchor protein which ensures the fixation of the multienzyme complex on the support.
5. The Peculiarities of Functioning Multienzyme Complexes One can expect that the multienzyme complex fixed on the biological support will function in close cooperation with the support. The interactions between the components of the complex must provide the coordination in catalytic action of the enzymes. It is evident that the usual approaches elaborated for analysis of kinetic behaviour of the systems with homogeneous distribution of the enzymes cannot be used for description of functioning of the multienzyme complex fixed on the support. The new approaches must be applied with this aim. One of the approaches may be based on the consideration of the enzyme complex states which differ in the degree of the completeness of catalytic process for individual enzymes of the complex. The interaction of the initial state of the complex with the first substrate of the metabolic pathway is bimolecular reaction. The following transformations of the complex may be considered as the monomolecular transitions which are accompanied by the transfer of the intermediates from one enzyme to the active site of the neighbouring enzyme. Such an approach was used, for example, for description of the electron
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transfer between structurally-bound carriers of the mitochondrial respiratory chain (Rubin & Shinkarev, 1984). Another approach which may be useful, in our opinion, for description of functioning of the multienzyme complex fixed on the support as a whole is based on the application of theory of autowaves (Zhabotinskii, 1974; Ivanitskii et al., 1978). The multienzyme complex may be considered as an excitable medium, each of whose elements (i.e. enzymes) is able to exist in one of three qualitatively different states: the state of rest (the enzyme contains the bound substrate and is ready to perform the catalytic act); the state of excitation (the performing of the catalytic act); and the state of refractoriness (the removal of the products of the reaction from the active site). The attachment of the substrate to the enzyme means the transition from the state of refractoriness into the state of rest. The wave of excitation in the multienzyme complex is initiated by binding of a corresponding chemical signal to the anchor protein of the support to which the multienzyme complex is attached. The passing of the excitation wave in the multienzyme complex initiates the metabolic process in the microcompartment formed by this complex. It should be noted that the properties of the autowave (the rate, the form of the profile, the amplitude) do not depend on the initial conditions initiating it and are determined by the features of the medium, i.e. by the catalytic characteristics of the enzymes, by the character of the interactions of the enzymes in the complex and by the influence of the metabolite-regulators bound to the sites located on the surface of the complex. The initial function of the enzymes in the multienzyme complex takes place in the region of excitation of the autowave. The order of functioning of the enzymes is determined by the structure of the multienzyme complex and the character of moving the autowave and may differ from the order of the positions of the enzymes in the metabolic pathway. The enzyme which directly interacts with the anchor protein of the support (i.e. 6-phosphofructokinase in the complex of glycolytic enzymes) is initiated in the first instance. The changes in the conformation of 6-phosphofructokinase molecule accompanying the catalytic act provoke the transition of neighbour enzymes (glucosephosphate isomerase, pyruvate kinase, fructose-bisphosphate aldolase, lactate dehydrogenase) in the state of excitation. Further extension of the autowave is exerted as a result of conformational changes of the enzymes proceeding to catalytic conversion of the substrates. The transfer of the intermediates on the conveyor takes place probably in the state of the refractoriness. The duration of the refractoriness state is longest for 6-phosphofructokinase which is characterized by the lowest turnover number among the glycolytic enzymes. One can assume that the passing of the excitation wave in the glycolytic enzymes complex fixed on structural proteins of skeletal muscle is coupled with sliding of actin and myosin filaments past each other. 6. The Role of Nature of the Support
It should be noted that the same multienzyme complex may be fixed on different supports. For example, the complex of glycolytic enzymes is formed on the thin filaments of I-band of muscle fibres and on the dimers of band 3 protein (the
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anion-transport system) embedded in the e~throcyte membrane (Green et al., 1965; Tillman et al., 1975; Kurganov, 1984; Kurganov et al., 1985). The attachment of the complex of glycolytic enzymes to the dimers of band 3 protein is shown in Fig. 1.
~
--
l lllllll II II IIIIIIl l l t t l RWgll gll !~, "~
l l l l Illll~U IlI~/WMNNIIJ ml
inside
II ~f IllntlllllllRIiiIlnllmulgllgR
UU u l s m ~ w m m ~ l t outside
FIG. 1. The schematic representation of the attachment of the complex of glycol~ic enzymes to the dimers of band 3 protein embedded in the erythrocyte membrane. Designations: 1 is band 3 protein, 2 is glucosephosphate isomerase, 3 is 6-phosphofructokinase, 4 is fructose-bisphosphate aldolase, 5 is glyceraldehyde-phosphate dehydrogenase, 6 is phosphoglycerate kinase, 7 is phosphoglyceromutase, 8 is enolase, 9 is pyruvate kinase, 10 is lactate dehydrogenase, 11 is triosephosphate isomerase and 12 is glycerol-3-phosphate dehydrogenase.
The nature of second messengers is the same in cells of different types. So the anchor proteins in different matrices are controlled by the same chemical signals. It is known that the band 3 protein is able to bind Ca 2÷ ions which have the inhibitor), effect on the transport of anions through the erythrocyte membrane (Passing & Schubert, 1983). One can expect that the functioning of the glycolytic complex fixed on dimers of band 3 protein will be sensitive to Ca 2÷ ions as in the case of the complex adsorbed to the thin filaments of the muscle fibres. It seems ve~ likely that the functioning of the multienzyme complexes fixed on the anchor proteins of different nature will have specific peculiarities. If the integral membrane-bound proteins play the role of the anchor sites, the formation of the multienzyme complex on the support and its functioning will be influenced by the composition, fluidity and phase state of lipid components of the membrane. The oligomeric state of the anchor protein, its ability to interact with the peripheral enzymes and the chemical signals depend on its microenvironment in the membrane. Many integral membrane-bound enzymes function as systems exerting the transport of certain metabolites through the membrane. The adsorption of the peripheral enzymes to membrane-bound protein channels results in the formation of a united "transport-metabolic system" which provides the functional linkage of metabolic processes proceeding in the cell compartments separated by the membrane. The channel-forming protein plays the role of the "control centre" of a similar system.
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When explaining the interactions of the metabolic systems one usually takes into account the regulatory relationships which are exerted with the participation of the common metabolites. In my opinion, such an approach is simplified. It is impossible to understand the interrelationships between the metabolic systems without eliciting the chemical signals regulating each of the metabolic systems as a whole. Consider, for example, two metabolic systems (M~ and M2) ensuring the energetic requirements of the cell. The coordinated functioning of both metabolic systems which provides the effective using of the degradable substrates and the stabilization of the levels of the metabolite-indicators of the cell energetic state may be exerted by influence of the chemical signals produced by the higher levels of the control of metabolism upon the anchor proteins of the supports. The nature of these anchor proteins (f/~ and 1/2 in Fig. 2) and the character of their interactions with the chemical signals (X~ and X2) are different. In order to switch on one of the metabolic system with simultaneous switching off of the other metabolic system the chemical signals must exist which are characterized by the opposite character of action on these metabolic systems. Each of the metabolic systems represented schematically in Fig. 2 has two types of signals one of which stimulates and the other suppresses the rate of a given metabolic pathway.
FIG.2. The schemewhichillustratesthe coordinatedfunctioningof two metabolicsystems(MI and M2) fixedto the anchor proteins of the support (f~ and f~2)-X~ and X~ represent the chemicalsignals whichregulatethe metabolicsystemsby the actionon the anchorproteinsof the supports (+ = activation, - = inhibition). It should be noted that the regulatory relations must exist which coordinate the development of the signals of the opposite types. It is known, for example, that the phosphorylation of membrane proteins catalyzed by cyclic AMP-dependent protein kinases causes the change in the permeability of the membrane with respect to Ca 2+ ions. On the other hand, Ca 2+ ions affect the activity of adenylate cyclase. Ca 2+ ions in relatively high concentration inhibit adenylate cyclase, but low concentrations of Ca 2+ ions have the activating effect. The second effect is mediated by calmodulin. The complex of Ca 2+ with calmodulin activates cyclic AMP phosphodiesterase which destroys cyclic AMP (Berridge, 1975; Rodbell, 1983; Tkachuk, 1983). One must take into account the feedbacks between a metabolic system (the object of the control) and the systems transforming the signals from the higher levels of
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the control of metabolism. The stimulation of a metabolic system by chemical signals interacting with the anchor protein of the support must inevitably cause the intensification of the processes directed to decreasing the power of incoming chemical signal. The stimulation of glycolytic process results in the enhancement of production of ATP which may be used by Ca2+-ATPase for pumping off Ca 2+ from sarcoplasm into the contents of the vesicles of sarcoplasmic net. One of the important problems of current metabolism is the underestanding of the control of futile cycle elements. As an example we can mention the cyclic interconversions between fructose-6-phosphate and fructose-l,6-bisphosphate. The phosphorylatio n of fructose-6-phosphate into fructose-l,6-bisphosphate is catalyzed by 6-phosphofructokinase (the enzyme of the glycofytic pathway). The hydrolysis of fructose-l,6-bisphosphate to fructose 6-phosphate is catalyzed by fructose-l,6bisphosphatase (the enzyme of gluconeogenic pathway). The cyclic interconversions between these substrates are accompanied by useless expenditure of ATP and therefore this cycle must be strictly controlled. On the basis of the fact of the complexation between 6-phosphofructokinase and fructose-l,6-bisphosphatase (Uyeda & Luby, 1974; Proffitt et al., 1976) one can propose the existence of the united complex of glycolytic and gluconeogenic enzymes. The effective regulation of the interconversions of fructose-6-phosphate and fructose-l,6-bisphosphate may be achieved with the participation of the anchor protein to which the complex is attached. It seems very likely that the coordinated proceeding the glycolytic and gluconeogenic pathways depends on the state of the anchor protein interacting with second messengers. In summary, we suppose that the rough coordination in functioning of the metabolic systems is exerted with the participation of the signals aitecting the "control centres" of these systems, the anchor proteins of the support (this is the "high" level of the control). The regulatory relations which are realized with the participation of the common metabolite-regulators ensure the fine coordination in functioning of the metabolic systems (the "low" level of the control). These levels of the control are characterized by difterent rates of response. In accordance with the modern data of the temporary organization of the living systems the time constant for each high level of the control is much greater than the analogous value for the lower level (Gadzhiev & Chernyshev, 1976).
8. Conclusion
The principles of the control of metabolic systems proposed in the present paper may be useful for the formulation of the general laws of integration of cellular metabolism. The progress in this field will be determined by obtaining the information about the presence of multienzyme complexes in the cell Only on the basis of similar information can one objectively select the "primary blocks" in cellular metabolism--the metabolic systems. The elucidation of the nature of the support which fixes the multienzyme complex gives the key to the understanding of the mechanisms which exert the switching on or switching off the metabolic system.
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I n m o d e r n b i o l o g i c a l c h e m i s t r y there is n o t a b r a n c h w h i c h d e a l s with the c h e m i c a l signals ( a n d o t h e r m a t e r i a l factors) w h i c h c o n t r o l certain m e t a b o l i c system as a whole. T h e m a j o r i t y o f such c o m p o u n d s ( C a 2÷ ions, cyclic A M P a n d others) are k n o w n b u t we d o n o t y e t realize their role as signals w h i c h switch o n o r switch off m e t a b o l i c systems. T h e e l a b o r a t i o n o f this b r a n c h o f b i o l o g i c a l c h e m i s t r y will m e a n the c o n s i d e r a b l e a d v a n c e in the u n d e r s t a n d i n g o f the m e c h a n i s m s o f the i n t e g r a t i o n of cellular metabolism. When constructing models of regulation of cellular metabolism, one must pay special a t t e n t i o n to the p r o b l e m o f s p a t i a l o r g a n i z a t i o n o f the m e t a b o l i c systems u n d e r study. The m o d e l s with h o m o g e n e o u s d i s t r i b u t i o n o f the e n z y m e s have l i m i t e d value. T h e t h e o r y o f i n t e g r a t i o n o f c e l l u l a r m e t a b o l i s m m u s t be e l a b o r a t e d on the basis o f t h e o r y o f m u l t i l e v e l h i e r a r c h i c a l systems a n d the t h e o r y o f the c o n t r o l h i e r a r c h y in s u c h systems ( W a t e r m a n , 1968; M e s a r o v i r , 1968; G a d z h i e v & C h e r n y shev, 1976; K o n e v , 1979).
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