The role of multienzyme complexes in integration of cellular metabolism

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 I-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 of the multienzyme complex (switching on-switching off the metabolic system). It is assumed that glycolytic system in skeletal muscles is stimulated by Ca2+ ions which interact with the anchor protein of the support (troponin C).     

Principles of integration of cell metabolism

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).     

Supramolecular organization of glycolytic enzymes

On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscle and to erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites the structure of glycolytic enzyme complex adsorbed to a biological support has been proposed. The key role in the formation of the multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex fixed on structural proteins of skeletal muscle contains one tetrameric molecule of 6-phosphofructokinase and at two molecules of other glycolytic enzymes. Hexokinase is not involved in the complex composition. The molecular mass of the multienzyme complex is about 2,6 X 10(6) Da. The formation of the multienzyme complex leads to the compartmentation of the glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.     

Supramolecular organization of glycolytic enzymes

On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscles and to the erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites, the structure of the glycolytic enzymes complex adsorbed to a biological support has been proposed. The key role in the formation of multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex contains one tetrameric molecule of 6-phosphofructokinase and two molecules of each of other glycolytic enzymes. Hexokinase is not a part of the complex. The molecular mass of the multienzyme complex is about 2.6 X 10(6) daltons. The multienzyme complex has symmetry axis of second order. The formation of the multienzyme complex leads to the compartmentation of glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.     

Adsorption of peripheral enzymes to membrane anchor proteins

The character of the isotherms of specific adsorption of peripheral enzymes to dimeric anchor proteins embedded in the membrane has been analysed. The situations are discussed when adsorption corresponds to the stoichiometry of one or two molecules of peripheral enzyme per dimeric binding site. The corresponding expressions describing the competitive interrelationships between peripheral enzymes adsorbed to the same binding sites have been derived. The experimental data on the adsorption of glycolytic enzymes to erythrocyte membranes are used for the illustration of the theoretical predictions. The physiological role of enzyme self-association which leads to the formation of enzyme oligomers of unlimited length is discussed. It is assumed that under in vivo conditions the association sites of such enzymes are saturated through interactions with anchor proteins of subcellular structures and with the enzymes of the corresponding metabolic pathways. Therefore the linearly associating enzymes play the key role in the formation of multienzyme complexes attached to subcellular structures. The significance of 6-phosphofructokinase adsorption to erythrocyte membranes in the formation of the complex of glycolytic enzymes is discussed.     

A broad view of scaffolding suggests that scaffolding proteins can actively control regulation and signaling of multienzyme complexes through allostery

Enzymes often work sequentially in pathways; and consecutive reaction steps are typically carried out by molecules associated in the same multienzyme complex. Localization confines the enzymes; anchors them; increases the effective concentration of substrates and products; and shortens pathway timescales; however, it does not explain enzyme coordination or pathway branching. Here, we distinguish between metabolic and signaling multienzyme complexes. We argue for a central role of scaffolding proteins in regulating multienzyme complexes signaling and suggest that metabolic multienzyme complexes are less dependent on scaffolding because they undergo conformational control through direct subunit–subunit contacts. In particular, we propose that scaffolding proteins have an essential function in controlling branching in signaling pathways. This new broadened definition of scaffolding proteins goes beyond cases such as the classic yeast mitogen-activated protein kinase Ste5 and encompasses proteins such as E3 ligases which lack active sites and work via allostery. With this definition, we classify the mechanisms of multienzyme complexes based on whether the substrates are transferred through the involvement of scaffolding proteins, and outline the functional merits to metabolic or signaling pathways. Overall, while co-localization topography helps multistep pathways non-specifically, allosteric regulation requires precise multienzyme organization and interactions and works via population shift, either through direct enzyme subunit–subunit interactions or through active involvement of scaffolding proteins. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Problems of biochemical organization]

Biological organization has been defined as a unity of structure, function and regulation. Biological organization of hierarchical multilevel biological systems is represented by a hierarchy of functioning controllable structures. The hierarchy of levels of material organization predetermines the existence of a hierarchy of regulatory mechanisms. Biochemical organization involves the levels of material organization corresponding to biomacromolecules, supramolecular complexes and cellular organelles. The levels of biomacromolecules and supramolecular structures effectuating elementary functions and controlled by basic regulatory mechanisms occupy key positions in biological systems. These levels play the role of standard functional blocks; their combination leads to hierarchically higher structural levels (cell, tissue, organ, systems of organs, organism) performing more complex functions and controlled by hierarchically more important regulatory mechanisms. The peculiarities of regulation of biological systems that are due to the existence of a hierarchy of regulatory mechanisms are discussed.     

The tentative identification in Escherichia coli of a multienzyme complex with glycolytic activity.

Penicillin spheroplasts of Escherichia coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20-30% of the glycolytic enzymes without increasing their specific activities. There was, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of five, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered. An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a molecular weight of about 1.6 X 10(6). Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 X 10(6), which contained a proportion of all the glycolytic enzyme studied; the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual molecular weight. The fraction of mol. wt 1 600 000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both the eluted and the reconstructed systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6-phosphate was reduced by the presence of unlabelled 3-phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates. Electron microscope investigation of both systems showed large numbers of regular 30 nm diameter particles which, on disruption, appeared to be composed of smaller units: it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.     

Organization of enzymes on subcellular structures: relay at the surface

There is a large body of evidence that soluble cytoplasmic enzymes of eukaryotic cells, e.g., glycolytic enzymes and proteins of the translational machinery, are organized in some way in space and in time. The following features of such organization emerge from the experimental data: (1) metabolites are transferred between enzymes directly "from hand to hand" in short-living enzyme-enzyme complexes rather than by diffusion in aqueous media; (2) enzymes show a tendency to be absorbed on surfaces of subcellular structures, such as membranes, cytoskeleton and polyribosomes; (3) enzymes are desorbed from a surface of a subcellular structure after binding specific metabolites, i.e., substrates and/or products of the reactions catalyzed by these enzymes. These features are suggestive of a relay mechanism for the enzyme systems functioning in a cell; an enzyme adsorbed on a surface of a subcellular structure is desorbed after binding its substrate or in the course of the catalytic act. Within a complex with its product the enzyme diffuses into the environment, until it reaches the next enzyme adsorbed on the same surface; then a short-living enzyme-enzyme complex is formed, and a direct "from hand to hand" transfer of the metabolite takes place. As a result, the overall metabolic process appears to be localized near the surface. We termed this mechanism as a "relay at the surface".     

Where is the glycolytic complex? A critical evaluation of present data from muscle tissue

Associations between glycolytic enzymes and subcellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzyme binding to subcellular structural components is believed to regulate enzyme activity in vivo through the formation of a multi-enzyme complex. However, three lines of evidence suggest that enzyme binding to cellular structures is not involved in the control of glycolysis. (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strength and higher enzyme concentrations indicate that a large multi-enzyme complex would not exist. (ii) In many cases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity. (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negligible.     

Mathematical analysis of multienzyme systems. II. Steady state and transient control.

The control theory of steady states, previously presented for linear enzymatic systems (Heinrich and Rapoport, 1974) is extended to nonlinear systems. On the basis of three theorems a new procedure for the calculation of the control strength and of the control matrix is developed. The theory is applied to the extended model of glycolysis of erythrocytes, which includes also ATP-consuming processes. Also in this model the glycolytic flux is mainly controlled by the hexokinase-phosphofructokinase-system. The control strengths of the pyruvate kinase and of the enzymes of the 2.3 P2G-bypass are negligibly small. The control strength of the ATPase is negative, i.e. an activation of this enzyme leads to a decrease of the flux. For transition states of multienzyme systems definitions are given for the mean time required for the transition of the metabolites and for the "transient control" of enzymes. Enzymes with a pronounced influence on the transition time are called time-limiting enzymes. Enzymes which excert strong control on the time-dependent processes may have little influence under steady state conditions and vice versa. The transition times of ATP have been calculated for transient states of glycolysis.     

Design of glycolysis

The design of the glycolytic pathway resulting from the continuous refinement of evolution is discussed with regard to three aspects. 1. Functional and structural properties of individual enzymes. The catalytic constants of the glycolytic enzymes are remarkably optimized; the turnover numbers are within one order of magnitude. The same is true for the molarities of catalytic centres in the cytosol, as is noted for yeast. Functional properties of the enzymes are reflected in their tertiary and quaternary structures. 2. Regulatory mechanisms of single enzymes. A classification of the various types of enzymic control mechanisms operating in the glycolytic pathway is given. In addition to the usual Michaelis-Menten saturation kinetics and the various types of inhibition there is control by positive and negative effectors based on oligomeric structures (fast acting, fine control) as well as regulation by chemical interconversion structures (fast acting, fine control) as well as regulation by chemical based on enzymes cascades (slow acting, very effective). 3. Functional and regulatory mechanisms of the whole glycolytic reaction pathway. A prominent feature is the high enzyme:substrate ratio, which guarantees fast response times. However, a quantitative treatment of the overall kinetics is limited by an incomplete knowledge of the enzymes' dynamic and chemical compartmentation as well as some of their control properties. From an analysis of the oscillatory state, certain control points in the glycolytic chain can be located that coincide with major branching points to other metabolic pathways. These points are controlled by fast-acting cooperative enzymes that operate in a flip-flop mechanism together with the respective antagonistic enzymes, preventing futile cycles. The gating enzymes leading to the glycogen store and the citric acid cycle are of the slow-acting but very effective interconvertible type. The combination of all the complex and intricate features of design yields a glycolytic network that enables the cell to respond to its various metabolic needs quickly, effectively and economically.     

The complex of band 3 protein of the human erythrocyte membrane and glyceraldehyde-3-phosphate dehydrogenase: stoichiometry and competition by aldolase

The cytoplasmic domain of band 3, the main intrinsic protein of the erythrocyte membrane, possesses binding sites for a variety of other proteins of the membrane and the cytoplasm, including the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase. We have studied the stoichiometry of the complexes of human band 3 protein and GAPDH and the competition by aldolase for the binding sites. In addition, we have tried to verify the existence of mixed band 3/GAPDH/aldolase complexes, which could represent the nucleus of a putative glycolytic multienzyme complex on the erythrocyte membrane. The technique applied was analytical ultracentrifugation, in particular sedimentation equilibrium analysis, on mixtures of detergent-solubilized band 3 and dye-labelled GAPDH, in part of the experiments supplemented by aldolase. The results obtained were analogous to those reported for the binding of hemoglobin, aldolase and band 4.1 to band 3: (1) the predominant or even sole band 3 oligomer forming the binding site is the tetramer. (2) The band 3 tetramer can bind up to four tetramers of GAPDH. (3) The band 3/GAPDH complexes are unstable. (4) Artificially stabilized band 3 dimers also represent GAPDH binding sites. In addition it was found that aldolase competes with GAPDH for binding to the band 3 tetramer, and that ternary complexes of band 3 tetramers, GAPDH and aldolase do exist.     

Principles of the organization and functioning of the metabolon microcompartment

The approaches to the elucidation of principles of metabolon functioning and the applicability of concepts of "protein-machine" and "conjugated ionic hydrogen bond systems" are discussed. The term "metabolon" refers to a supramolecular complex of sequential metabolic enzymes and structural elements of the cell. It has been assumed that the metabolon microcompartment consists of separate units joined together by channels, in which the active and, possibly, allosteric sites of enzymes become approximated.     

Mechanisms of regulation and interplastid localization of chlorophyll biosynthesis.

The current concepts of chlorophyll biosynthesis, its interplastid localization, biosynthetic and biochemical heterogeneity, mechanisms of regulation of the key reactions, formation of 5-aminolevulinic acid and incorporation of magnesium into protoporphyrin IX, are reviewed. The literature and author's data demonstrate the existence of in vivo multienzyme systems synthesizing chlorophyll and its precursors as monovinyl and divinyl chemical species. Both types of the multienzyme systems synthesize 5-aminolevulinic acid and regulate this process independently. A hypothesis is considered that the function of the magnesium branch of chlorophyll biosynthesis in vivo is controlled by a mechanism through inhibition of the enzymes by their products because of the limitation of the binding sites for them in the membrane. An additional influence of light on the Mg-chelatase activity not only via the photosynthetic supply with ATP but also through the light-induced synthesis of the enzyme molecules de novo is described. Efficient energy migration from protoporphyrin IX and Mg-protoporphyrin IX (monomethyl ester) molecules to the protochlorophyllide active form detected by the author is discussed considering a close location of these pigments in plastid membranes and the enzymes participating in their formation.     

The association of glycolytic enzymes with cellular and model membranes

This article deals with the binding of glycolytic enzymes with membranous or protein subcellular structures. The representative papers of the last three decades dealing with this matter are reviewed. The studies evidencing the binding of some glycolytic enzymes to insoluble subcellular proteins and membranous structures are presented. It is currently generally accepted that the glycolytic enzymes work in some organisation. Such organisation undoubtedly plays a marked role, although still poorly known, in the regulation processes of glycolysis. From this review, the conclusion emerges that the regulatory ability of the binding of glycolytic enzymes to cellular membranes should be added to the list of well-known mechanisms of post-translational regulation of the glycolytic enzymes. Some of the results presented are the background for the hypothesis that planar phospholipid domains in/on the membrane surface are capable of functioning as binding sites for these enzymes. Such binding can modify the conformation state of the enzymes, which results in changes in their kinetic properties; thus, it may function as a regulator of catalytic activity     

Mechanism of 1,3-bisphosphoglycerate transfer from phosphoglycerate kinase to glyceraldehyde-3-phosphate dehydrogenase

1. The kinetics of 1,3-bisphosphoglycerate binding to glyceraldehyde-3-phosphate dehydrogenase have been examined by stopped-flow techniques in the absence and presence of phosphoglycerate kinase, using enzyme concentrations in the range 0.5-40 microM. Rate and equilibrium constant estimates for the interaction of the ligand with the two enzymes are reported. 2. The kinetics of ligand transfer from the binary complex of bisphosphoglycerate and phosphoglycerate kinase to the binary complex of NAD+ and glyceraldehyde-3-phosphate dehydrogenase conform excellently to the predictions of a standard free-diffusion mechanism and exhibit no detectable contributions from a mechanism of direct (channelized) transfer of bisphosphoglycerate between the two enzymes. 3. Previously reported evidence that the binary complex of bisphosphoglycerate and phosphoglycerate kinase may act (in the presence of NADH) as a substrate for glyceraldehyde-3-phosphate dehydrogenase according to Michaelis-Menten kinetics is based on a misinterpretation of the experimental observations that can be attributed to neglect of the autocatalytic effect of NAD+ produced during the reaction. Experiments performed under conditions where the autocatalytic effect of NAD+ is eliminated provide clear evidence that the kinetics of utilization of the kinase-bisphosphoglycerate complex for enzymic NADH reduction are consistent with prior dissociation of the complex according to a free-diffusion mechanism of metabolite transfer and incompatible with a mechanism of direct metabolite transfer. 4. A kinetic argument is presented which renders implausible the very idea that direct metabolite transfer between 'soluble' consecutive enzymes in metabolic pathways may offer any catalytic advantages in comparison to metabolite transfer by free diffusion. A mechanism of direct metabolite transfer seems intuitively attractive only because one tends to disregard the diffusional processes required to bring the consecutive enzymes together and to separate them when the transfer has been completed. Direct metabolite transfer would be expected to be catalytically advantageous only in tightly bound multienzyme complexes showing no kinetically significant tendency to dissociate. 5. It is concluded that mechanisms of direct metabolite transfer have not been convincingly demonstrated to apply, nor are they likely to apply, between 'soluble' consecutive enzymes in metabolic pathways, at least not in the glycolytic sequence of reactions.     

Equilibrium partition studies of the myofibrillar interactions of glycolytic enzymes

The interactions of several glycolytic enzymes with muscle myofibrils in imidazole-chloride buffer (pH 6.8, I 0.158) have been investigated by equilibrium partition studies. Results for aldolase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and phosphofructokinase are interpreted in terms of a myofibrillar capacity of 76 nmol/g protein and a single intrinsic association constant for each tetravalent enzyme with matrix sites. The existence of separate myofibrillar sites for aldolase and glyceraldehyde-3-phosphate dehydrogenase is established by demonstrating independence of the binding of each enzyme upon the presence of the other. Although this investigation provides further physicochemical support for myofibrillar adsorption of glycolytic enzymes in the cellular environment, its findings are incompatible with the proposition (B. I. Kurganov, N. P. Sugrobova, and L. S. Mil'man (1985) J. Theor. Biol. 116, 509-526) that the phenomenon reflects the formation of a specific multienzyme complex attached to the myofibril.     

The glycine decarboxylase system: a fascinating complex

The mitochondrial glycine decarboxylase multienzyme system, connected to serine hydroxymethyltransferase through a soluble pool of tetrahydrofolate, consists of four different component enzymes, the P-, H-, T- and L-proteins. In a multi-step reaction, it catalyses the rapid destruction of glycine molecules flooding out of the peroxisomes during the course of photorespiration. In green leaves, this multienzyme system is present at tremendously high concentrations within the mitochondrial matrix. The structure, mechanism and biogenesis of glycine decarboxylase are discussed. In the catalytic cycle of glycine decarboxylase, emphasis is given to the lipoate-dependent H-protein that plays a pivotal role, acting as a mobile substrate that commutes successively between the other three proteins. Plant mitochondria possess all the necessary enzymatic equipment for de novo synthesis of tetrahydrofolate and lipoic acid, serving as cofactors for glycine decarboxylase and serine hydroxymethyltransferase functioning.     

Engineering the spatial organization of metabolic enzymes: mimicking nature's synergy

A growing body of evidence indicates that many cellular reactions within metabolic pathways are catalyzed not by free-floating 'soluble' enzymes, but via one or more membrane-associated multienzyme complexes. This type of macromolecular organization has important implications for the overall efficiency, specificity, and regulation of metabolic pathways. An ever-increasing number of biochemical and genetic studies on primary and secondary metabolism have laid a solid foundation for this model, providing compelling evidence in favor of the so-called channeling of intermediates between enzyme active sites and colocalization of enzymes inside a cell. In this review, we discuss several of nature's most notable multifunctional enzyme systems including the AROM complex and tryptophan synthase, each of which provides new fundamental insights into the structural organization of metabolic machinery within living cells. We then focus on the growing body of literature related to engineering strategies using protein chimeras and post-translational assembly mechanisms. Common among these techniques is the desire to mimic natural enzyme organization for optimizing the production of valuable metabolites with industrial and medical importance.