BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E. S. Severin - 2004

SECTION 2. ENZYMOLOGY

VII. Regulation of Metabolic Processes

A living Cell is an open system that continuously exchanges matter and energy with its environment. It takes up nutrients, which undergo transformations and serve as structural and energetic building blocks, while metabolic end products are excreted from The Cell. In a multicellular Organism, a cell responds not only to environmental changes but also to the functional activity of neighboring Cells, all the while striving to maintain a constant internal composition. This state is known as stationary state or METABOLISM/37.html">Cellular Homeostasis.

The cell constantly hosts a vast array of Chemical Reactions that form metabolic pathways—the sequential conversion of certain compounds into others. Metabolism is the sum total of all metabolic pathways occurring within the cells of an organism.

Among all Metabolic Pathways in the organism, two opposing processes are distinguished: Catabolism and anabolism. Catabolism involves The breakdown of complex substances into simpler ones, accompanied by the release of energy. Anabolism is the synthesis of more complex substances from simpler ones. Metabolic pathways are coordinated in terms of Location, timing, and rate. This precise coordination of all processes is ensured by complex and diverse regulatory mechanisms.

A. Organization of chemical Reactions into Metabolic Pathways

The optimal activity of Enzymes catalyzing the reactions of a single metabolic pathway is achieved through a specific spatial organization within the cell.

1. Spatial Localization of Enzymes

Most enzymes are intracellularly localized and distributed unevenly throughout the organism. As a rule, all enzymes of a particular metabolic pathway reside in the same cellular compartment. Compartmentalization of metabolic pathways is especially crucial for opposing Catabolic and anabolic processes. For instance, fatty acid synthesis occurs in the Cytoplasm, whereas their breakdown takes place in Mitochondria. Without such Separation, functionally and energetically futile pathways would arise.

In metabolic pathways, the product of the first enzymatic reaction serves as the substrate for the second, and so on, until the end product is formed. Intermediates of a metabolic pathway can be withdrawn from the reaction sequence and utilized in other pathways; in other words, metabolic pathways are interconnected through their intermediate products.

In some cases, the Spatial Organization of enzymes is so pronounced that a reaction product cannot under any circumstances be isolated from the metabolic pathway and must necessarily serve as the substrate for the next reaction. Such an arrangement of a metabolic pathway is known as a multienzyme complex, which arises from the Structural and functional organization of enzymes. Typically, these complexes are membrane-bound. Examples of multienzyme complexes include the Pyruvate dehydrogenase complex, which drives The oxidative decarboxylation of pyruvic acid (pyruvate) (see Section 6), and fatty acid synthase, which catalyzes the synthesis of palmitic acid (see Section 8).

2. Structure of Metabolic Pathways

The structure of metabolic pathways within the cell is extremely diverse (see Table 2-3). When a substrate is converted into a single product through a series of enzymatic steps, such a pathway is termed a linear metabolic pathway. Branched metabolic pathways are also common, leading to the synthesis of various end products depending on the cell's needs. As you study biological chemistry, you will also encounter cyclic and spiral metabolic pathways.

Class="center">Table 2-3. Types of Metabolic Pathways

Organ Specificity

The enzymatic composition of different cells varies. Housekeeping enzymes, which perform essential life-sustaining Functions, are found in all Cells of the organism. During cellular differentiation, the enzymatic profile of cells undergoes changes. For example, the enzyme arginase, involved in urea synthesis, is found exclusively in Liver cells, whereas acid phosphatase, which participates in the Hydrolysis of orthophosphoric acid monoesters, is located in prostate cells. These are referred to as organ-specific enzymes.

When it comes to highly specialized cells, they contain a higher Abundance of enzymes tailored to their specific functions compared to other cell types. For instance, Heart Muscle cells contain elevated levels of creatine kinase and aspartate aminotransferase, liver cells are rich in Alanine aminotransferase and aspartate aminotransferase, and osteoblasts have high concentrations of alkaline phosphatase, and so forth.

Compartmentalization

The cell is a sophisticated functional system that regulates its own maintenance. The multitude of cellular functions is ensured by spatial and temporal Regulation of Specific metabolic pathways (primarily dependent on nutritional rhythms). Spatial regulation relies on the strict localization of specific enzymes within various Organelles. Thus, The Nucleus houses enzymes associated with DNA and RNA Synthesis, the cytoplasm contains glycolytic enzymes, Lysosomes contain hydrolytic enzymes, the mitochondrial matrix contains TCA cycle enzymes, and The inner mitochondrial membrane holds the Electron Transport Chain enzymes, and so on (Fig. 2-29). Such subcellular localization of enzymes promotes the orderly progression of biochemical processes and enhances metabolic rates.

Fig. 2-29. Intracellular Localization of Enzymes.

B. Principles of Regulation of metabolic Pathways

All chemical reactions in the cell proceed with the participation of enzymes. Therefore, to influence The rate of a metabolic pathway, it is sufficient to regulate the quantity or activity of these enzymes. Typically, metabolic pathways feature Key Enzymes that govern the rate of the entire pathway. These enzymes (one or more per pathway) are known as regulatory enzymes; as a rule, they catalyze the initial steps of a metabolic pathway, irreversible reactions, rate-limiting reactions (the slowest ones), or reactions situated at pathway branch points (metabolic switches).

The Rate of Enzymatic reactions is regulated at 3 independent levels:

✵ by changing the number of enzyme molecules;

✵ through the availability of substrate and coenzyme molecules;

✵ by altering the catalytic activity of the enzyme molecule.

1. Regulation of the number of enzyme molecules in the cell

It is known that cellular Proteins undergo constant turnover. The number of enzyme molecules in a cell is determined by the balance between two processes—the Synthesis and degradation of the enzyme protein molecule:

Protein Synthesis AND folding represent a multistep process. The Regulation of Protein Synthesis can occur at any stage of protein molecule formation. The best-studied mechanism of protein synthesis regulation is at the transcriptional level, which is mediated by specific metabolites, Hormones, and various biologically active molecules (see Section 4).

Regarding enzyme degradation, The regulation of this process is less understood. One can only assume that this is not merely a simple proteolysis process (the breakdown of a protein molecule), but rather a complex mechanism, likely determined at the genetic level.

2. REGULATION OF ENZYMATIC reaction rate via substrate and coenzyme availability

An important parameter controlling the flux of a metabolic pathway is the availability of substrates, primarily the initial substrate. The higher the concentration of the starting substrate, the greater the rate of the metabolic pathway.

Another parameter that limits the flow of a metabolic pathway is the availability of regenerated Coenzymes. For example, in dehydrogenation reactions, the oxidized forms of NAD⁺, FAD, and FMN serve as coenzymes for dehydrogenases and are reduced during the reaction. For these coenzymes to participate in the reaction again, their regeneration is required, i.e., their conversion back into the oxidized form.

3. Introduction/15.html">Regulation of enzyme catalytic activity

Of paramount importance in altering the rates of metabolic pathways is the regulation of the catalytic activity of one or more key enzymes within that pathway. This is a highly efficient and rapid method of Metabolic Regulation.

The MAIN MECHANISMS OF ENZYME ACTIVITY REGULATION include:

✵ Allosteric Regulation;

✵ regulation via Protein-Protein Interactions;

✵ regulation through phosphorylation/dephosphorylation of the enzyme molecule;

✵ regulation by partial (limited) proteolysis.

Allosteric regulation

Allosteric enzymes are those whose activity is regulated not only by the concentration of substrate molecules, but also by other substances known as effectors. The effectors involved in allosteric regulation are cellular metabolites, often originating from the very pathway whose activity they modulate.

The Role of allosteric enzymes in cellular metabolism. Allosteric enzymes play a vital role in metabolism because they respond extremely rapidly to the slightest Changes in the cell's internal environment. Allosteric regulation is of great importance in the following situations:

✵ in anabolic processes. Feedback inhibition by the end product of a metabolic pathway and activation by initial metabolites enable precise regulation of the synthesis of these compounds;

✵ in catabolic processes. When ATP accumulates in the cell, it inhibits metabolic pathways responsible for energy production. Consequently, substrates are redirected toward reactions that store energy reserves;

✵ for the coordination of anabolic and Catabolic pathways. ATP and ADP act as allosteric effectors that function as antagonists;

✵ to coordinate parallel and interrelated metabolic pathways (e.g., the Synthesis of purine and pyrimidine NUCLEOTIDES used for nucleic acid synthesis). Thus, the End products of one metabolic pathway can act as allosteric effectors of another.

Allosteric effectors. An effector that causes a decrease (inhibition) in enzyme activity is called a negative effector, or inhibitor. An effector that causes an increase (activation) in enzyme activity is called a positive effector, or activator.

Various metabolites frequently serve as allosteric effectors. The end products of a metabolic pathway are often inhibitors of allosteric enzymes, whereas starting Materials act as activators. This phenomenon is known as heterotropic regulation, a type of allosteric control that is widespread in biological systems.

A rarer case of allosteric regulation occurs when the substrate itself acts as a positive effector. This is termed homotropic regulation (where the effector and substrate are the same substance). These enzymes possess Multiple binding sites for the substrate that can perform a dual function: catalytic and regulatory. Such allosteric enzymes operate when a substrate accumulates in excess and must be rapidly converted into product.

Allosteric Enzymes can be identified by studying their kinetics. They do not obey Michaelis–Menten kinetics and exhibit a characteristic sigmoidal (S-shaped) curve when reaction velocity is plotted against Substrate Concentration.

Structural and functional features of allosteric enzymes:

✵ they are typically Oligomeric Proteins composed of several protomers or featuring a domain structure;

✵ they possess an allosteric site spatially distinct from the catalytic Active Site;

✵ effectors bind non-covalently to the enzyme at allosteric (regulatory) sites;

✵ like catalytic sites, allosteric sites can exhibit varying specificity toward ligands, ranging from absolute to group specificity. Some enzymes contain multiple allosteric sites, some specific for activators and others for inhibitors.

✵ the protomer bearing the allosteric site is termed the regulatory protomer, as opposed to the catalytic protomer, which contains the active site where the chemical reaction takes place;

✵ allosteric enzymes exhibit cooperativity: the binding of an allosteric effector to its site induces sequential, Cooperative conformational changes across all subunits, altering the conformation of the active site and modifying the enzyme's affinity for the substrate, which consequently decreases or increases catalytic activity (Fig. 2-30);

Fig. 2-30. Diagram illustrating The Mechanism of an allosteric enzyme. A — action of a negative effector (inhibitor); B — action of a positive effector (activator).

✵ allosteric regulation is reversible: dissociation of the effector from the regulatory subunit restores the enzyme's initial catalytic activity;

✵ allosteric enzymes catalyze the key committed steps of a given metabolic pathway.

Localization of allosteric enzymes within a metabolic pathway. The rate of metabolic processes depends on the concentration of the substances consumed and produced in a given reaction chain. This regulation is logical, because when an end product accumulates, it can act as an allosteric inhibitor of the enzyme that typically catalyzes the initial step of that metabolic pathway:

The enzyme catalyzing The conversion of substrate A into product B contains an allosteric site for a negative effector, which is the end product of the metabolic pathway, F. If the concentration of F increases (i.e., substance F is synthesized faster than it is consumed), The activity of one of the early enzymes is inhibited. This type of regulation is known as negative feedback or feedback inhibition. Negative feedback is a common mechanism for regulating cellular metabolism.

In central metabolic pathways, initial substrates can act as activators of key pathway enzymes. As a rule, allosteric activation typically targets enzymes that catalyze the key Reactions of the final stages of a metabolic pathway:

As an example, we can examine the regulatory principles of Glycolysis, the specific (initial) pathway of glucose degradation (Fig. 2-31). One of the end products of glucose breakdown is the ATP molecule. An excess of ATP in the cell triggers feedback inhibition of the allosteric enzymes Phosphofructokinase and pyruvate kinase. Conversely, the accumulation of high levels of fructose-1,6-bisphosphate leads to the allosteric activation of pyruvate kinase.

Fig. 2-31. Scheme of positive and negative REGULATION OF GLUCOSE catabolism. ATP molecules participate in the feedback inhibition of the allosteric enzymes phosphofructokinase and pyruvate kinase. Fructose-1,6-bisphosphate acts as an activator of the glucose degradation pathway. Plus signs indicate activation, and minus signs indicate Enzyme Inhibition.

This type of regulation ensures the smooth and coordinated progression of the glucose degradation pathway.

Regulation of enzyme catalytic activity by protein-protein interactions. Some enzymes alter their catalytic activity As a result of protein-protein interactions. Let us examine two MECHANISMS OF ENZYME activation mediated by protein-protein interactions:

✵ enzyme activation resulting from the binding of regulatory proteins;

✵ changes in the catalytic activity of enzymes due to the association or dissociation of enzyme protomers.

Enzyme activation by regulatory protein binding. This type of regulation can be illustrated by the activation of the enzyme adenylate cyclase, which is localized in The Plasma Membrane of the cell.

The Active Site of adenylate cyclase is located on the cytoplasmic face of the plasma membrane. Activated adenylate cyclase catalyzes the conversion of ATP into cyclic 3',5'-AMP (cAMP)—a secondary intracellular messenger of hormone action (see the diagram below).

Within the membrane, adenylate cyclase functions in a complex with other proteins:

✵ a hormone receptor exposed to the extracellular environment that interacts with hormones;

✵ a G-protein, which occupies an intermediate position between the receptor and the enzyme adenylate cyclase. The G-protein is an oligomeric protein consisting of 3 subunits: α, β, and γ. The α-subunit contains a GTP-binding and Cleavage site; hence, this protein is referred to as a GTP-binding protein, or G-protein;

✵ hormone binding to the receptor induces a conformational change in the G-protein, decreasing its affinity for the GDP molecule (to which it is bound in the absence of a hormonal signal) and increasing its affinity for GTP. The attachment of GTP induces Conformational Changes in the G-protein and its dissociation into subunits: the GTP-bound α-subunit (α-GTP) and the βγ dimer;

✵ α-GTP has a high affinity for adenylate cyclase, and its binding leads to the activation of the latter. Therefore, α-GTP acts as a regulatory protein, and this

mechanism of adenylate cyclase activation is referred to as enzyme activation via regulatory protein binding (Fig. 2-32).

Fig. 2-32. Regulation of adenylate cyclase activity. A hormone (H), interacting with a receptor (R) on the cell surface, leads to a decrease in the affinity of the GTP-binding protein (G-protein, consisting of α, β, and γ protomers) for GDP and an increase in its affinity for GTP. The binding of a GTP molecule to the active site of the G-protein causes the dissociation of the complex into the α-GTP subunit and the βγ dimer. The α-GTP complex activates adenylate cyclase, which promotes the synthesis of intracellular regulatory molecules of cAMP from ATP. AC – adenylate cyclase, PKA – protein kinase A, R_i – H₃PO₄.

Regulation of enzyme catalytic activity by association/dissociation of protomers

Protein Kinases — a group of enzymes that catalyze The transfer of a phosphoric acid residue from ATP to specific OH groups of amino acid residues in proteins (causing protein phosphorylation). The activation mechanisms of various protein kinases differ. As an example of regulating enzyme catalytic activity through the association or dissociation of protomers, we can consider the regulation of protein kinase A activity.

Protein kinase A (cAMP-dependent) consists of 4 subunits of 2 types: 2 regulatory (R) and 2 catalytic (C). Such a tetramer lacks catalytic activity. The Regulatory Subunits possess binding sites for cyclic 3',5'-AMP (cAMP), two per subunit. The binding of 4 cAMP molecules to the 2 regulatory subunits induces a conformational change in the regulatory protomers and the dissociation of the tetrameric complex, thereby releasing 2 active catalytic subunits (Fig. 2-32). This regulatory mechanism is reversible. The dissociation of cAMP molecules from the regulatory subunits leads to the association of the regulatory and catalytic subunits of protein kinase A, forming an inactive complex.

Regulation of enzyme catalytic activity via phosphorylation/dephosphorylation

In biological systems, the Regulation of Enzyme Activity through the Covalent Modification of amino acid residues is a frequent occurrence. A rapid and widespread method of chemical modification of enzymes is phosphorylation/dephosphorylation. The OH groups of the enzyme are subjected to modification. Phosphorylation is carried out by protein kinases, whereas dephosphorylation is mediated by phosphoprotein Phosphatases. The attachment of a phosphoric acid residue alters the conformation of the active site and its catalytic activity. The outcome can be twofold: Some enzymes are activated upon phosphorylation, whereas others, conversely, become less active (Fig. 2-33).

Fig. 2-33. Regulation of enzyme activity by phosphorylation/dephosphorylation.

The change in enzyme activity caused by phosphorylation is reversible. The removal of the phosphoric acid residue is carried out by phosphoprotein phosphatases. The activity of protein kinases and phosphoprotein phosphatases is regulated by hormones, which allows for rapid modulation of key metabolic pathway enzymes in response to environmental conditions. Functionally antagonistic hormones exert opposite effects on the phosphorylation/dephosphorylation of enzymes, leading to contrasting shifts in cellular metabolism.

For instance, under The Influence of Glucagon (between meals), cells exhibit a decrease in the synthesis of energy reserves—fat and Glycogen—and an enhancement of their breakdown (mobilization) triggered by the phosphorylation of key enzymes involved in these processes. Conversely, under the influence of Insulin (during Digestion), glycogen synthesis is activated and its breakdown is inhibited, as the interaction of insulin with its receptor triggers a signaling pathway leading to the dephosphorylation of those same key enzymes.

Regulation of enzyme catalytic activity by partial (limited) proteolysis

Certain enzymes that function outside cells (in the gastrointestinal tract or Blood Plasma) are synthesized as inactive precursors and are activated only through the hydrolysis of one or more specific peptide bonds, which results in the cleavage of a portion of the precursor protein molecule. As a result, a conformational rearrangement occurs in the remaining part of the protein molecule, forming the active site of the enzyme.

Let us consider the mechanism of Limited proteolysis using the activation of the proteolytic enzyme Trypsin as an example (Fig. 2-34). Trypsinogen, synthesized in the Pancreas, travels through the pancreatic ducts during digestion into the duodenum, where it is activated via limited proteolysis by the intestinal enzyme enteropeptidase. The cleavage of an N-terminal hexapeptide leads to The formation of the active site within the remaining part of the molecule. It is worth recalling that trypsin belongs to the family of Serine proteases, meaning its active site contains a functionally crucial Ser residue.

Fig. 2-34. Activation of trypsin by limited proteolysis. The intestinal enzyme enteropeptidase catalyzes the hydrolysis of the Lys-Ile peptide bond. The Cleavage of the N-terminal hexapeptide results in the Formation of the active site in the remainder of the protein molecule.

Limited proteolysis is an example of regulation where enzyme activity is altered irreversibly. Such enzymes typically function for a short duration, determined by the lifespan of the protein molecule. Limited proteolysis underlies the activation of Proteolytic Enzymes, proteins involved in Blood Coagulation and Fibrinolysis, Complement system proteins, as well as Peptide Hormones.