Fundamentals of Biochemistry - Filippovich, Y. B. 1999

Interrelationship and Regulation of Metabolism
Regulation of Metabolism

The data presented above on the interconnection and interdependence of protein, nucleic acid, carbohydrate, and Lipid METABOLISM demonstrate that metabolism is a harmonious ensemble of numerous, tightly coordinated chemical processes. Protein bodies play the leading role in this countless multitude of interactions. Thanks to their catalytic function, this vast array of chemical degradation and synthesis processes is carried out. Nucleic Acids maintain strict Specificity during The Biosynthesis of macromolecules, i.e., ultimately, species specificity in The Structure of key Biopolymers. Through carbohydrate and lipid metabolism, the reserves of ATP—the universal energy donor for chemical transformations—are continuously replenished in the Organism. These same substances supply the simplest organic molecules used to build biopolymers and Other Compounds. As a result, a continuous process of self-renewal of living matter takes place, driven by those biochemical mechanisms whose study is the subject of general biochemistry.

The coordination of biochemical transformations, their close connection and interdependence, the possibility of rapidly mobilizing some compounds to synthesize others, the potential for mutual transitions from one Class of Organic compounds to another, and the universal subordination of biochemical mechanisms stand out more clearly than ever when we evaluate Metabolism as a whole. The general course of biochemical processes in the organism, regulated by internal and external factors, represents a single, inseparable whole, and the organism itself in this sense appears as a self-tuning, self-regulating system that maintains its own existence through metabolism.

Nevertheless, for methodological reasons, it is customary to examine The regulation of life processes at the metabolite, Operon, cellular, organismal, and population levels. Each of these is characterized by its own regularities of Metabolic Regulation, which are integrated and preserved at each subsequent level of the Organization of living matter. Characteristically, the higher the level of metabolic regulation, the more evident the hierarchical, block-based control system becomes, The Essence of which boils down to monitoring signal input and responding to it.

Metabolite level of regulation. The coordination of Metabolism in the Organism is largely determined by the concentration of various metabolites—low-molecular-weight compounds that are products of certain chemical transformations in biological objects or enter them through Nutrition.

The forms of metabolic regulation involving metabolites are extremely diverse. The simplest of them comes down to accelerating or slowing down biochemical processes due to a deficiency or excess of those compounds that participate in the corresponding reactions. Thus, the volume of Protein Synthesis in heterotrophs is limited by the intake of Essential Amino Acids and the intensity of semi-essential Amino acid synthesis. This principle forms the basis, in particular, for the microbiological method of Quantitative determination of amino acid content in protein hydrolysates and other media.

A more complex character is typical of metabolic regulation through competitive interactions between metabolic processes that converge on common metabolites, which generally belong to the category of Key Intermediates: pyruvic, oxaloacetic, and a-ketoglutaric acids, acetyl-CoA, and glucose-6-phosphate. Numerous Examples of this kind are provided in the section on metabolic interconnection at the beginning of this chapter.

Undoubtedly, a major role in the Regulation of Metabolic processes is played by A number of low-molecular-weight compounds classified as BIOLOGICALLY ACTIVE SUBSTANCES—Vitamins, Antivitamins, Coenzymes, HORMONES, antihormones, second messengers, etc.

Metabolites, by interacting with Enzymes, are capable of activating or inhibiting their activity; an example of the former is the repeatedly mentioned activation of protein Kinases upon the action of cAMP on them. Another second messenger plays an equally important role in metabolic regulation—cGMP, which activates phospholipases A₂ and C and is also involved in the biosynthesis of Prostaglandins from arachidonic acid. As recently shown, phosphoinositides serve as the source of a new group of second messengers (see p. 384):

The hormonal signal is perceived by a receptor that transmits it to a G-protein, which activates phospholipase C. Both diacylglycerol and Inositol-1,4,5-trisphosphate act as second messengers that transmit the hormonal signal further: the former to protein kinase C (M = 67–83 kDa, activated by Ca²⁺, transfers phosphate from ATP to Serine and Threonine residues, comprises at least 7 forms—a, ßI, ßII, y, δ, ε, ζ, phosphorylating about 20 enzymes and a number of Proteins such as Cytoskeleton and receptor proteins), and the latter to The Endoplasmic reticulum, from which Ca²⁺ is released, stimulating The activity of Ca²⁺/calmodulin-dependent protein kinase, which also ensures the phosphorylation of functionally significant proteins (Fig. 139). Nitric oxide (NO)—a potent Homeostasis factor—also belongs to the second messengers. It is formed in various Cells and Tissues, including human platelets, from L-Arginine via The oxidation of the nitrogen of the amino group in the guanidine moiety by the action of L-arginine-NO synthase. It exists in three forms, two of which are constitutive and one is inducible, with lipopolysaccharides and a nuclear protein factor being involved in the Induction of the latter's Gene Expression. The nitric oxide thus generated activates guanylate cyclase, leading to an increased concentration of cGMP, whose Functions in metabolic regulation were noted above (see the previous page). In addition, nitric oxide itself directly acts as a neurotransmitter and cytotoxic agent.

An example of the latter is feedback inhibition (retroinhibition) of the activity of an enzyme located at the beginning of a multistep substrate conversion by the final reaction product, a mechanism that has been worked out in detail in The Study of Pyrimidine Nucleotide Biosynthesis regulation (see p. 239):

Fig. 139. Phosphoinositide pathway of metabolic regulation (explained in the text)

Similar phenomena occur in many other cases of allosteric enzyme regulation. At the same time, metabolites can also act as isosteric (competitive) Enzyme Inhibitors (see p. 112). It has been suggested that metabolons (see p. 355) are regulated by signals transmitted via second messengers; for instance, it is believed that the glycolytic metabolon is regulated by an influx of Ca ions into the microcompartments of The Cell where it is localized. Finally, metabolites function as Inducers and co-repressors in systems operating at the operon level of metabolic regulation.

Operon level of regulation. An operon is an ordered, compact cluster of cistrons (along with start and stop signals) transcribed as a single unit during mRNA synthesis on a DNA template. In the case of a monocistronic operon, it synthesizes an mRNA molecule dedicated to the biosynthesis of a single specific protein in the cell's ribosomal machinery; in the case of a polycistronic operon (containing up to a dozen or so cistrons), it yields a set of mRNAs that direct the ribosomal assembly of a family of different proteins (most commonly enzymes) required for a multistep biochemical process within the cell. A foundational Overview of the monocistronic operon, as well as its Transcription start and stop signals, is provided in Fig. 87 on p. 259.

It is now well established that the operon level primarily regulates The rate of enzyme biosynthesis by modulating the number of mRNA molecules generated during transcription. This exerts a decisive influence on metabolic pathways, which are heavily driven by enzymes. However, one must not overlook the fact that the operon level also regulates mRNA synthesis for the de novo formation of Histones, non-histone and ribosomal proteins, and various other proteins that lack catalytic activity yet serve as regulators of The Genome's metabolic activity, the cellular translational machinery, and other fundamental metabolic processes.

Fig. 140. Operon model

A closer look at the operon-level Introduction/15.html">Regulation of enzyme biosynthesis reveals two distinct pathways: Induction and Repression. The core principles of both are illustrated in Fig. 140. Enzyme biosynthesis can be induced by a low-molecular-weight metabolite—an inducer—which binds to a repressor protein (normally blocking transcription), thereby freeing the operator region (the operon's start signal). This allows RNA polymerase to bind and initiate the synthesis of pro-mRNA, which is subsequently processed into the mature enzyme following post-transcriptional modification.

Enzymes whose biosynthesis is regulated in this manner are termed inducible. These include ß-galactosidase, ribulokinase, tyrosinase, asparaginase, and many others. The addition of an inducer (typically the substrate of the inducible enzyme) dramatically increases the rate of enzyme synthesis. For example, adding a ß-galactoside (lactose) to an Escherichia coli culture medium increases ß-galactosidase synthesis 10,000-fold. In contrast, enzymes whose biosynthesis is halted by a low-molecular-weight metabolite known as a co-repressor (Fig. 140) (which converts a normally non-repressor protein into an active form capable of occupying the operator region) are called repressible. Examples include Ornithine carbamoyltransferase (whose co-repressor is arginine), Glutamine Synthetase, and urease (whose co-repressor is NH₄), among others.

Recent studies have demonstrated that induction and repression can be generalized—that is, controlled not by a single specific inducer or co-repressor, but by an entire group of structurally similar compounds. Accumulating evidence also suggests that a single effector (e.g., ppGpp — 3'-pyrophosphoguanosine-5'-diphosphate) can trigger the biosynthesis of an entire family of enzymes (such as those involved in the biosynthesis of Histidine and several Other Amino Acids). Consequently, the MECHANISMS OF ENZYME induction and repression are highly complex, particularly regarding the induction of enzymes foreign to a given organism (e.g., insecticide detoxification enzymes in insects).

Naturally, the REGULATION OF METABOLISM at the cellular genetic level is not restricted solely to the induction and repression of enzyme synthesis. Both DNA Replication and the transcription of various RNAs (including mRNAs) from a DNA template—which largely dictate the course of cellular metabolism—depend on a multitude of additional events. These include DNA Methylation; the phosphorylation and Acetylation of histones and non-histone proteins within Chromatin; the interaction of hormone-receptor complexes with chromatin; and the adenylylation of proteins involved in the replication machinery, among others. All of these processes are linked to shifts in the METABOLIC ACTIVITY OF the genome and the regulation of its overall functions.

Cellular level of regulation. Regulatory processes at THE CELLULAR LEVEL include: nuclear-cytoplasmic interactions; post-transcriptional and post-translational modifications of macromolecules; the Transport of substances across the membranes of subcellular particles and the endoplasmic reticulum; macromolecular interactions (protein-protein, protein-nucleic acid, carbohydrate-protein, and lipid-protein); and others. Each of these plays a fundamental role in metabolic regulation.

Nuclear-cytoplasmic interactions boil down to the interdependent control over the synthesis of vital, functionally active biopolymers. For instance, the small protein subunits of ribulose-1,5-bisphosphate carboxylase—which drives the crucial process of CO₂ fixation in plant cells (see p. 360)—are synthesized in the Cytoplasm, whereas the large subunits are synthesized in the Chloroplasts. Consequently, the biosynthesis of the former is directed by the cellular nuclear genome, while that of the latter is governed by the Chloroplast Genome localized within the cytoplasm. Overall, of the 800–1,000 proteins required for chloroplast function, only about 15% are encoded by the genome of these cellular Organelles. In addition to ribulose-1,5-bisphosphate carboxylase, the assembly of chloroplast thylakoid membranes, ATPase complexes, and RNA polymerase complexes relies on the cooperative efforts of both The plant cell's nuclear and chloroplast genetic systems. A similar nuclear-cytoplasmic coordination regulates the synthesis of protein subunits for other vital catalytic systems, such as proton-translocating ATPase and cytochrome c oxidase, Proteins of the inner and outer mitochondrial membranes, and proteins of chloroplast and mitochondrial Ribosomes. Thus, complex protein-enzyme cellular complexes—which sustain fundamental metabolic pathways—arise solely through the coordinated activity of the nuclear genome alongside the genomes of Mitochondria, chloroplasts, and other subcellular structures, driven by the synchronized operation of the protein-synthesizing systems within these compartments.

Posttranscriptional and posttranslational modification of macromolecules is the second most critical regulatory process at the cellular level. Ribonucleic acid precursors generated during transcription undergo a series of transformations—such as methylation, Cleavage, and the addition of oligonucleotide fragments—to become functionally active RNAs. These processes have been studied in detail during the maturation of mRNA, rRNA, and tRNA (see Chapter VI). Overall, they determine the intensity of protein synthesis within the cell. However, proteins produced via ribosomal synthesis also undergo posttranslational modification, including methylation, cleavage of peptide fragments, and the addition of carbohydrate moieties during glycoprotein biosynthesis. Consequently, precursor Polypeptides yield active enzymes, hormones, biologically active Peptides, and other substances. Naturally, many metabolic processes in the cell depend directly or indirectly on the extent of posttranslational modification.

In recent years, particular importance has been ascribed to the Covalent Modification of enzymes, as this mechanism regulates the activity of at least 100 of them. In addition to phosphorylation at serine, threonine, and Tyrosine residues by corresponding protein kinases (see Chapters IX and XII), adenylylation, uridylylation, and ADP-ribosylation of enzymes play major roles.

Adenylylation and uridylylation, for instance, have been studied in detail by E. Stadtman in glutamine synthetase:

Deadenylylated glutamine synthetase is active in the biosynthesis of glutamine; as adenylic acid residues are added to each of its 12 subunits (potentially allowing for 382 different enzyme forms), its activity decreases. Adenylylation occurs at the tyrosine residue:

In turn, adenylyl transferase is active only when bound to a regulatory protein that is not uridylylated at the tyrosine residue. If the regulatory protein is uridylylated (in a reaction analogous to adenylylation, but utilizing UTP as the donor of the uridylate residue), adenylyl transferase accelerates the deadenylylation of glutamine synthetase, returning it to a more active state.

In the case of ADP-ribosylation, an ADP-ribose moiety from $\text{NAD}^+$ is transferred to the guanidino group of an enzyme's arginine residue (or to the $\text{H}_2\text{N}$ group of Lysine or asparagine), releasing a nicotinamide molecule. This reaction is accelerated by $\text{NAD}^+$ase, which exhibits ADP-ribosyltransferase activity:

Closely linked to posttranslational protein modification is proteolytic protein degradation, particularly of enzymes. This ensures a continuous antagonism within the cell between two opposing processes—enzyme breakdown and synthesis—which ultimately determines The amount of a given enzyme and the rate of the reaction it catalyzes.

Posttranslational modification is a source of multiple enzyme forms within the cell, as seen, for example, in glutamine synthetase. Along with genetically determined isozymes, they provide the finest nuances in the regulation of metabolic processes, with each functioning like a distinct instrument in an orchestra whose combined sound forms the symphony of life. Recently, researchers have also been actively exploring the control of nuclear and Mitochondrial Genome transcription by cytoplasmic products in animals and plants.

The transport of substances across the membranes of The Nucleus, mitochondria, Lysosomes, endoplasmic reticulum, and other subcellular elements, as well as through The Cell wall, is a vital mechanism for regulating metabolism and numerous physiological Functions of the organism at the cellular level. Cyclic peptides and depsipeptides play a leading role in ion transport, whereas low-molecular-weight substances are transported by specialized enzymes (translocases) (see Fig. 43). High-molecular-weight compounds, particularly proteins, are transported via signal peptides and their interactions with the protein-Lipid Components of membranes. A distinct category of proteins known as porins has recently been discovered and studied: they form pores in membranes and actively transport various compounds through them (see Figs. 101 and 102).

Fig. 141. Protein-Nucleic Acid Interactions during mRNA maturation and transport.

Increasing attention is being devoted to substance transport processes in membranology. Ultimately, The entry of metabolites into subcellular particles and cellular compartments, as well as their efflux, directly affects the rate of biochemical transformations. Of course, The problem of the Spatial Compartmentalization of metabolic reactions within the cell is not limited solely to the creation of metabolite concentration gradients. Current research focuses on particle-bound enzymes, where enzymatic activity increases significantly (the "adsorption mechanism" of enzyme regulation, accompanied by the Relay-like transfer of catalytic intermediates).

The Role of macromolecular interactions in regulating cellular metabolism became particularly evident through the study of protein-nucleic acid interactions. It was found that pre-mRNAs synthesized in the nucleus initially bind to Nuclear Proteins (informofers), and Selection of pre-mRNAs occurs at this stage: only a fraction of them mature into ribonucleoproteins (30S particles and their assemblies), while the rest, currently non-essential for the metabolic needs of the cell, are degraded. Within 30S particles, pre-mRNAs mature and translocate across the nuclear envelope, shedding their protein component in the process. However, upon entering the cytoplasm, the mRNAs rebind to high-affinity cytoplasmic particles to form informosomes. Within informosomes, mRNAs remain in a latent state until they reach the ribosomes. Clearly, this cascade mechanism of information transfer from the nucleus to the cytoplasm has a profound impact on metabolism (Fig. 141). Research in this field, conducted at our institution under the supervision of Academics G. P. Georgiev and A. S. Spirin, was awarded the USSR State Prize. Diverse and crucial protein-nucleic acid interactions regulate metabolism at the genetic and ribosomal levels of the cell.

Over the past decade, fundamentally new data have emerged regarding Metabolic control at the level of protein-nucleic acid interactions. It has been discovered that the primary role of the universal and most thoroughly studied eukaryotic protein kinase—specifically, casein kinase type II—consists in regulating the masking and unmasking of mRNA by interacting proteins. In this context, both the mRNA molecules themselves and a distinct class of other RNAs act as regulators of protein kinase activity: the more heavily phosphorylated the RNA-binding proteins are, the lower their affinity for mRNA, and the greater the mRNA's capacity to participate in translational processes.

Equally impressive results have been obtained in the study of Lipid-Protein Interactions. Particularly important for metabolic regulation are those occurring within the protein-lipid membranes of the cell. These interactions determine the activity levels of membrane-bound enzymes, membrane permeability to metabolites, the transmembrane Transport of Macromolecules, and various other processes dependent on the physicochemical parameters of the membrane apparatus.

The study of carbohydrate-protein interactions has opened a new chapter in our understanding of specific contacts at the level of macromolecules, subcellular particles, and cells. These interactions rely on proteins (Lectins) that selectively recognize carbohydrate components, thereby triggering processes critical for metabolic regulation.

As for Protein-Protein Interactions, their significance for metabolic Regulation at the cellular level is paramount. They involve the assembly of multimeric enzymes, multi-enzyme complexes, enzyme assemblies and metabolons, and hormone-receptor complexes in the case of Peptide and Protein hormones. Specific examples of these phenomena were discussed previously.

Until recently, virtually no data existed on biologically significant carbohydrate-carbohydrate interactions. Today, the situation is diametrically opposed. The principles of intercellular adhesion mediated or initiated by carbohydrate-carbohydrate recognition have been uncovered, with the latter serving as a rapid primary process preceding protein-protein adhesion. Researchers have established the characteristics of contacts within the membrane apparatus, where the density of embedded Glycolipids is high, and have obtained striking data regarding the functional roles and regulatory potential of these molecules.

Organismal Level of regulation. The primary mechanism of metabolic regulation at the organismal level is hormonal (see Chapter XII). Operating via humoral pathways in animals and through conducting tissue systems in plants, this regulation is, in turn, directed by Nervous system signals in the former and environmental cues in the latter. Thus, this represents a natural and logical transition from biochemistry to physiology.

Populational level of regulation. Like the preceding level, this tier of regulation lies at the interface of biochemistry and physiology, gradually evolving into a new science: chemical ecology. Consequently, it is now more accurate to speak of the level of metabolic regulation in ecosystems, keeping in mind the global aspects of chemical interactions in living nature. Its essence boils down to the potent influence exerted by chemical compounds produced and released by certain individuals on the metabolism and behavioral responses of others. This is mediated through receptor systems or target Tissues of the recipient organism. Relevant examples concerning Antibiotics and telergones were mentioned earlier (see Chapter IV). However, the range of substances participating in intra- and interspecific chemical interactions among individuals is far broader and continuously expanding. These include phytoncides—antibacterial substances produced by healthy plants (the research of B. P. Tokin and his students played a major role in their study); phytoalexins—protective compounds synthesized by plants in response to bacterial or fungal infection; and novel types of antibiotics, Plant Hormones, neurohormones, and so forth. Comprehensive investigation and deep understanding of The Nature and mechanisms of these biochemical relationships in the wild are crucial for developing the ecological strategies so urgently needed by humanity today.