BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 7. ENZYMOLOGY

7.7. Regulation of Metabolic Processes

7.7.3. Main Mechanisms of Enzyme Catalytic Activity Regulation

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 typically cellular metabolites belonging to the very metabolic pathway they regulate.

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Allosteric enzymes play a vital role in METABOLISM because they respond extremely rapidly to the slightest Changes in the internal state of The Cell. Allosteric regulation is particularly important in the following situations:

✵ during anabolic processes. End-product inhibition of a metabolic pathway and activation by initial metabolites allow for precise control over the synthesis of these compounds;

✵ during catabolic processes. Accumulation of ATP within the cell inhibits energy-yielding metabolic pathways, thereby directing substrates toward energy storage and reserve reactions;

✵ for the coordination of anabolic and Catabolic pathways, where ATP and ADP act as antagonistic allosteric effectors;

✵ for coordinating parallel and interconnected metabolic pathways (such as the Synthesis of purine and pyrimidine NUCLEOTIDES used in nucleic acid synthesis). Thus, the End products of one metabolic pathway can serve as allosteric effectors for another.

An effector that decreases (inhibits) enzyme activity is called a negative effector, or inhibitor, whereas one that increases (activates) enzyme activity is called a positive effector, or activator.

Various metabolites most commonly act as allosteric effectors. The end products of a metabolic pathway often function as inhibitors of allosteric enzymes, while starting Materials act as activators. This phenomenon is known as heterotropic regulation and is widespread in biological systems.

Less commonly, allosteric regulation occurs when the substrate itself acts as a positive effector. This type of regulation is termed homotropic (where the effector and substrate are the same substance). Such enzymes possess multiple substrate-binding sites capable of performing a dual function: catalytic and regulatory. Allosteric enzymes of this type operate when a substrate accumulates in excess and needs to be rapidly converted into product.

Allosteric Enzymes can be identified through kinetic studies. These enzymes do not obey Michaelis-Menten kinetics; instead, they exhibit a characteristic sigmoidal (S-shaped) curve for the dependence of reaction velocity on Substrate Concentration.

Structural and functional features of allosteric enzymes:

✵ they are typically Oligomeric Proteins composed of multiple protomers or possessing a domain Structure;

✵ they feature an allosteric site spatially distinct from the catalytically Active Site;

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

✵ like catalytic sites, allosteric sites can exhibit varying degrees of Ligand Specificity, ranging from absolute to group specificity. Some enzymes possess multiple allosteric sites, with some specific to activators and others to inhibitors;

✵ the protomer bearing the allosteric site Functions as 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 a sequential, cooperative conformational change across all subunits. This alters the conformation of the active site and modifies the enzyme's affinity for the substrate, thereby increasing or decreasing catalytic activity (Fig. 7.30);

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

✵ allosteric enzymes catalyze key, rate-limiting reactions of a given metabolic pathway.

The rate of metabolic processes depends on the concentration of substances consumed and produced within a given reaction chain. This type of 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 features an allosteric site for a negative effector, which is the end product of metabolism G. If the concentration of G increases (i.e., substance G is synthesized faster than it is consumed), The activity of one of the initial enzymes is inhibited. This type of regulation is known as negative feedback, or

retroinhibition. Negative feedback is a widespread mechanism for regulating cellular metabolism.

Fig. 7.30. Diagram illustrating the function of an allosteric enzyme:

A - action of a negative effector (inhibitor); B - action of a positive effector (activator); АлЦ - allosteric site; АЦ - active site (catalytic); I - inhibitor; S - substrate; А - activator; 1 - binding of the inhibitor to the allosteric site; 2 - decrease in the affinity of the active site for the substrate, reduction in catalytic activity; 3 - binding of the activator to the allosteric site; 4 - increase in affinity for the substrate, enhancement of catalytic activity

In central metabolic pathways, starting materials can act as activators of Key Enzymes within the pathway. As a rule, allosteric activation typically targets enzymes that catalyze the final reactions of a metabolic pathway:

As an example, let us examine the regulatory principle of Glycolysis, the specific (initial) pathway of glucose degradation (Fig. 7.31). One of the end products of this degradation is an ATP molecule. When ATP is present in excess within the cell, retroinhibition of the allosteric enzymes Phosphofructokinase and Pyruvate kinase takes place. Conversely, The production of large amounts of fructose-1,6-bisphosphate leads to the allosteric activation of pyruvate kinase. Such regulation ensures the coordinated progression of glucose catabolic pathways.

Fig. 7.31. Diagram of positive and negative REGULATION OF GLUCOSE Catabolism:

Plus signs indicate enzyme activation, minus signs indicate Enzyme Inhibition

Regulation via Protein-Protein Interactions. Certain enzymes alter their catalytic activity As a result of such interactions. Let us consider two MECHANISMS OF ENZYME activation mediated by protein-protein interactions:

✵ enzyme activation resulting from the binding of regulatory proteins;

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

Enzyme activation through the binding of regulatory proteins. This type of regulation is exemplified by the activation of adenylate cyclase, an enzyme localized in the cell's Plasma Membrane.

The Active Site of adenylate cyclase is located on the cytoplasmic face of The Plasma Membrane. Activated adenylate cyclase catalyzes The formation of cyclic 3',5'-AMP (cAMP) from ATP, which acts as a secondary, intracellular messenger for hormonal action:

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

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

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

✵ upon hormone binding to the receptor, a conformational change occurs in the G protein, leading to a decreased affinity for the GDP molecule (to which it is bound in the absence of a hormonal signal) and an increased affinity for GTP. The binding of GTP induces Conformational Changes in the G protein, causing it to dissociate into subunits: the GTP-bound α-subunit (α-GTP) and the βγ dimer;

✵ α-GTP exhibits a high affinity for adenylate cyclase, and its binding activates the enzyme. Therefore, α-GTP acts as a regulatory protein, and this mechanism of adenylate cyclase activation is termed enzyme activation via the binding of regulatory proteins (Fig. 7.32).

Regulation of Catalytic activity through the association/dissociation of protomers. Protein Kinases are a group of enzymes that catalyze The transfer of a phosphate group from ATP to specific OH groups of protein amino acid residues (inducing protein phosphorylation). The activation mechanisms of various protein kinases are not identical. As an example of regulating catalytic activity via the association or dissociation of protomers, we can examine the Regulation of Protein kinase A activity.

Protein kinase A (cAMP-dependent) consists of four subunits of two types: two regulatory (R) and two catalytic (C). This tetramer lacks catalytic activity. The Regulatory Subunits possess binding sites for cyclic 3',5'-AMP (cAMP), two per subunit. The binding of four cAMP molecules to the two regulatory subunits induces a conformational change in the regulatory protomers and the dissociation of the tetrameric complex, thereby releasing two active catalytic subunits (Fig. 7.32). This regulatory mechanism is reversible. The dissociation of cAMP molecules from the regulatory subunits triggers the reassociation of the regulatory and catalytic subunits of protein kinase A to form the inactive complex.

Introduction/15.html">Regulation of enzyme catalytic activity through phosphorylation / dephosphorylation. In biological systems, enzyme activity is frequently regulated via the Covalent Modification of amino acid residues. A rapid and widespread method of chemical modification of enzymes is phosphorylation / dephosphorylation, which targets the -OH groups of the enzyme. Phosphorylation is catalyzed by protein kinases, whereas dephosphorylation is carried out 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, while others, conversely, become less active (Fig. 7.33).

Fig. 7.32. Regulation of adenylate cyclase activity:

hormone (H), by interacting with the receptor (R) on the cell surface, leads to a decrease in the affinity of the pyrimidine-binding protein (G-protein, consisting of α, β, γ protomers) for GDP and an increase in 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 cAMP from ATP. AC – adenylate cyclase, PKA – protein kinase A, Pi-H₃PO₄

Fig. 7.33. Regulation of Enzyme Activity by phosphorylation / dephosphorylation

Changes in enzyme activity induced by phosphorylation are reversible. The Cleavage of the phosphoric acid residue is performed by phosphoprotein phosphatases. The activity of protein kinases and phosphoprotein phosphatases is regulated by hormones, allowing for a rapid adjustment of key metabolic pathway enzymes in response to environmental changes. Antagonistically functioning hormones exert opposite effects on the phosphorylation / dephosphorylation of enzymes, leading to contrasting metabolic alterations within the cell. For instance, under the action 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 in these processes. Conversely, under The Influence of Insulin (during Digestion), glycogen synthesis is activated and its breakdown is inhibited, as insulin-receptor interaction triggers a signaling pathway that induces the dephosphorylation of those same key enzymes.

Regulation of enzyme catalytic activity through 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 upon the Hydrolysis of one or more specific peptide bonds, resulting in the cleavage of a portion of the precursor protein molecule. As a consequence, a conformational rearrangement takes place in the remaining part of the molecule, forming the enzyme's active site.

Let us examine The Mechanism of partial proteolysis using the activation of the proteolytic enzyme Trypsin as an example (Fig. 7.34). Trypsinogen, synthesized in the Pancreas, enters the duodenum via the pancreatic ducts during digestion, where it is activated via partial proteolysis by the intestinal enzyme enteropeptidase. As a result of the cleavage of the hexapeptide from the N-terminus, an active site is formed in the free region of the molecule. It is worth recalling that trypsin belongs to the family of "Serine proteases"—the active site of the enzyme contains a functionally important Ser.

Fig. 7.34. Activation of trypsin by partial proteolysis. The action of the intestinal enzyme enteropeptidase leads to the hydrolysis of the Lys—Ile peptide bond. As a result of the cleavage of the hexapeptide from the N-terminus, an active site is formed in the free region of the molecule

Partial proteolysis is an example of regulation whereby enzyme activity is altered irreversibly. The functional lifespan of such enzymes is typically short and is determined by the half-life of the protein molecule. Partial proteolysis underlies the activation of Proteolytic Enzymes, blood clotting and Fibrinolysis system proteins, Complement system proteins, as well as Peptide Hormones.