Textbook - BIOLOGICAL CHEMISTRY - Hubsky Yu.I. - 2000

Chapter II. GENERAL LAWS OF METABOLISM

CHAPTER 7. ENZYMES II. MECHANISMS OF CATALYSIS. KINETICS. REGULATION

7.3. REGULATION OF ENZYMATIC PROCESSES. ENZYMOPATHIES

An orderly course of biochemical reactions in the body is possible only in the presence of catalytically active forms of Enzymes at the right time and in the right place (in a tissue, Cell, or subcellular compartment), which requires a precise regulatory system (control) over enzyme activity. In addition, any Changes in the external or internal environment (such as diet, or the intensity of biological, chemical, and physical factors acting on the body) necessitate targeted adaptive responses of various physiological systems, which are realized through changes in the rates of specific metabolic processes catalyzed by enzymes.

There are two fundamental ways to regulate the intensity or rate of biochemical enzymatic reactions:

A — by altering the catalytic activity of the enzyme.

B — by altering The amount of the enzyme (or enzymes) that determine the course of the enzymatic process.

A. The first pathway of regulation involves the presence of special regulatory enzymes within The Cell's enzyme pool, usually located at the main, key junctions of METABOLISM. This pathway ensures the rapid adaptation of the body's enzymatic apparatus and takes place within a few seconds or minutes — representing a "rapid response" mechanism.

There are four main mechanisms for regulating the catalytic activity of enzymes (L. Stryer, 1995):

1. Allosteric Introduction/15.html">Regulation of enzyme Activity.

2. Regulation of enzyme activity through covalent modification.

3. Activation of enzymes via Limited proteolysis.

4. Activation and inhibition of enzyme activities by specific regulatory Proteins.

Class="center">Allosteric Enzymes

Allosteric enzymes are a type of regulatory enzyme that, In addition to the Active Site, possess an additional regulatory (allosteric) site with which Allosteric regulators (effectors, modulators) interact.

Allosteric effectors can be either positive, meaning they increase the catalytic activity of the enzyme (allosteric activators), or negative, meaning they inhibit it (allosteric inhibitors).

In terms of their molecular Structure, allosteric regulatory enzymes typically consist of multiple polypeptide chains, meaning they possess a quaternary structure. The active and regulatory (allosteric) sites are localized on different protein subunits — the catalytic and regulatory subunits, respectively. The Modification of the catalytic activity of such an enzyme is carried out by transmitting conformational changes from the Regulatory Subunits to the catalytic subunits, which occur in the former following interaction with ligands, i.e., effectors.

According to the model proposed by J. Monod, J. Wyman, and J.-P. Changeux (1965) (Fig. 7.11), there are two physical states of an allosteric enzyme that differ in their conformation and catalytic activity: the catalytic (relaxed) state (R-state) and the inhibited (tensed) state (T-state) (Fig. 7.11).

Fig. 7.11. Transition between the catalytically active (R) and inactive (T) forms of an allosteric enzyme (Monod-Wyman-Changeux model). C and R denote the catalytic and regulatory subunits, respectively.

The reverse transition between the R and T states depends on the interaction of the enzyme with allosteric effectors (activator or inhibitor, respectively), which, by interacting with binding sites on the regulatory subunit of the enzyme, stabilize its molecule in one of the conformational states.

Kinetics of Allosteric Enzymes

The kinetics of allosteric enzymes has A number of differences from the kinetics of conventional enzymes. The curve representing the dependence of reaction rate on Substrate Concentration for these enzymes is not hyperbolic, but rather S-shaped (sigmoidal). This is determined by cooperative effects of interaction between individual subunits of the regulatory enzyme during the binding of substrate molecules: at low concentrations, the substrate is practically unconverted, and only after the concentration reaches a certain threshold value does the reaction rate begin to increase rapidly (Fig. 7.12).

Fig. 7.12. Effect of Substrate concentration on reaction velocity for a standard (1 — hyperbolic curve) and an allosteric (2 — sigmoidal curve) enzyme.

Allosteric enzymes catalyze biochemical reactions that typically occur at the beginning of unbranched or branched metabolic pathways. The modulators of these Enzymes can be either their own substrates, known as homotropic regulatory enzymes, or other chemical effectors, particularly the End products of multi-step biochemical processes, known as heterotropic regulatory enzymes. In the latter case, the product (or products) of the metabolic pathway (usually of an anabolic, biosynthetic nature) inhibits The activity of the first enzyme in the sequence via a negative feedback mechanism, a process known as feedback inhibition:

A classic example of an enzyme regulated by an allosteric mechanism is aspartate transcarbamylase (ATCase), the first enzyme in the biosynthetic pathway for Pyrimidines, which are synthesized as nucleoside triphosphates such as uridine triphosphate (UTP) and cytidine triphosphate (CTP):

Given The Role of pyrimidines as components of DNA and RNA NUCLEOTIDES as well as certain Coenzymes, The regulation of ATCase catalytic activity is of paramount biological significance. Consequently, There is a precise control system governing their Abundance, which operates through the action of allosteric effectors on the activity of the pathway's initial enzyme, ATCase:

CTP, the end product of the biosynthetic pathway, acts as an allosteric inhibitor of ATCase, converting the enzyme into its inactive T-state;

ATP, a product of Purine Biosynthesis and an indicator of a high cellular energy state, Functions as an allosteric activator of the enzyme, shifting it into the active R-state.

In terms of its molecular architecture, ATCase is a typical regulatory enzyme composed of six regulatory and six catalytic subunits (R₆C₆) that form a protein with quaternary structure (Fig. 7.13).

Fig. 7.13. Quaternary Structure of aspartate transcarbamylase — top view. C — catalytic subunits, R — regulatory subunits.

The kinetics of substrate concentration dependence for the ATCase reaction are illustrated in Fig. 7.14. The addition of an allosteric activator (ATP) shifts the kinetic curve to the left, while an inhibitor (CTP) shifts it to the right.

Fig. 7.14. Kinetic curves of ACTase in the presence of ATP and CTP.

Covalent Modification of enzymes

Post-synthetic covalent modification of enzyme proteins is one of the common mechanisms for controlling metabolic processes. Pathways of such modification include reversible phosphorylation-dephosphorylation (the most widespread regulatory mechanism), as well as methylation, adenylylation, and ADP-ribosylation of enzyme proteins.

Proteins are phosphorylated by specialized enzymes known as protein Kinases (protein phosphokinases), which transfer the terminal (γ-) phosphate group from ATP to the Serine or Threonine (and in some protein kinases, Tyrosine) residues of the target protein:

The reverse reaction, protein dephosphorylation, is catalyzed by protein Phosphatases:

Substrates for protein kinases include numerous enzyme proteins (Glycogen phosphorylase, phosphorylase b kinase, glycogen synthase, triglyceride lipase, Pyruvate dehydrogenase, acetyl-CoA carboxylase, etc.), certain membrane channel proteins, Chromatin Histones, and more.

Phosphorylation of many enzyme proteins converts them into a catalytically active form (such as the phosphorylation of glycogen phosphorylase, phosphorylase b kinase, and triglyceride lipase); conversely, phosphorylation of other enzyme proteins (glycogen synthase, β-HMG-CoA reductase) serves as a mechanism for their inactivation. The Rate of protein phosphorylation-dephosphorylation typically ranges from a few seconds to a few minutes, according to the cell's need to turn a specific physiological function on or off.

A unique protein kinase—activated by cyclic adenosine monophosphate (cAMP), a secondary messenger in the cellular action of numerous physiologically active compounds (see below)—is involved in the regulation of numerous biochemical functions and physiological processes.

Proteolytic Enzyme Activation

Enzyme activation through limited proteolysis of their molecules is a mechanism that irreversibly transforms an enzyme into a catalytically active state. Under this mechanism, a specific peptide chain is cleaved from the inactive precursor form of the enzyme (proenzyme, zymogen); the remaining peptide undergoes conformational changes following limited proteolysis, leading to The formation of the active site and the Generation of the catalytically active enzyme protein.

This regulatory mechanism functions during the formation of active forms of most Proteolytic Enzymes in the digestive tract—Pepsin, Trypsin, and Chymotrypsin—as well as active proteases that are components (factors) of the human Blood Coagulation and fibrinolytic systems.

Action of Regulatory Proteins

The activity of certain proteins is controlled by specialized regulatory proteins that can exert activating or inhibitory effects.

Examples of such effector proteins include:

- calmodulin (CaM), a Ca-sensitive protein acting as a chemical sensor that translates an increase in cytosolic Ca²⁺ concentration into specific biochemical and physiological cellular responses; upon binding four Calcium Ions, the CaM-4Ca²⁺ complex becomes capable of activating numerous protein enzymes, notably cyclic nucleotide phosphodiesterase, Myosin light-chain kinase, and others;

- proteinase inhibitors, which are effectors that restrict (block) the activity of tissue proteinases—enzymes capable of degrading the body's own proteins; the most active inhibitors are α₂-macroglobulin and α₁-antitrypsin (α₁-proteinase inhibitor), which block the activity of serine and other proteinases by binding to their active sites;

- antihemophilic globulin A (blood coagulation factor VIII); this protein participates in the activation of factor X, which triggers the entire coagulation cascade leading to the formation of a blood clot; a hereditary deficiency of antihemophilic globulin A manifests as a bleeding tendency known as hemophilia.

B. The second regulatory pathway is a mechanism of long-term adaptation of the enzymatic apparatus. Its activation and full realization require several hours or days. In most cases, it involves changes in the biosynthesis rate of a specific enzyme protein through effects on the nuclear genome system or ribosomal Protein Synthesis (i.e., Transcription and Translation Processes). In certain biochemical systems, the amount of the enzyme protein within the cell increases by stabilizing existing molecules through the inhibition of the activity of proteases that degrade them.

This type of regulation is widespread in microorganisms, which exhibit a remarkable ability to adapt to changes in The chemical composition of the culture medium (concentrations of Amino Acids, CARBOHYDRATES, presence of specific Antibiotics, etc.) via the rapid activation or inhibition of the synthesis of corresponding enzymes.

Two classes of microbial enzymes are distinguished:

- constitutive enzymes, which are synthesized by bacterial Cells continuously, regardless of changes in environmental conditions;

- adaptive enzymes, whose biosynthesis rate varies depending on changes in environmental conditions.

Adaptive microbial enzymes are subdivided into inducible and repressible, meaning that their synthesis activity increases or decreases, respectively, in response to the action of specific effector compounds. The MOLECULAR MECHANISMS OF enzyme Induction and Repression in the bacterium E. coli will be discussed in Chapter 22.

Adaptive enzyme induction or repression also occurs in Eukaryotic cells. Examples of the induction of enzyme systems in The Human Body include changes in the concentration of Liver enzyme proteins occurring in response to the amount of dietary nutrients (adaptation of carbohydrate, amino acid, and Lipid Metabolism enzymes) and the intake of foreign compounds such as drugs or toxins (induction of detoxification enzymes, including glucuronidation and microsomal oxidation).

Cyclic Nucleotides in the Regulation of Enzymatic Processes

An important and widespread biological control system for enzymatic reactions, combining various molecular regulatory mechanisms, is the cyclic nucleotide system.

Cyclic nucleotides 3',5'-AMP (cAMP) and 3',5'-GMP (cGMP) are internal (3',5') diphosphoric esters of adenylic (AMP) and guanylic (GMP) acids.

The most widespread are cAMP-dependent control systems governing intracellular biochemical processes, particularly those subject to neurohumoral regulation by the whole Organism, which is mediated by Hormones and Neurotransmitters (Chapter 23). The regulation of enzymatic processes involving cAMP encompasses several successive stages of chemical (regulatory) signal Transduction and transformation.

1. The formation of cyclic nucleotides in Reactions Catalyzed by cyclase enzymes—adenylyl cyclase and guanylyl cyclase—from the nucleoside triphosphates ATP and GTP, respectively:

The Cleavage of cAMP and cGMP into regular, non-cyclic nucleoside monophosphates is catalyzed by cyclic nucleotide phosphodiesterase.

The enzyme adenylyl cyclase is located in the Plasma Membranes of cells, and its activation occurs As a result of interaction with Membrane Receptors for certain physiologically active compounds, notably hormones such as adrenaline, Glucagon, and others.

2. The activation of protein kinases by cyclic AMP, whose function is the phosphorylation of other protein enzymes. These cAMP-dependent protein kinases are regulatory enzymes activated by cAMP via an allosteric control mechanism.

cAMP-dependent protein kinase is a tetramer consisting of two catalytic and two regulatory subunits (C₂R₂), much like Other Enzymes with allosteric regulatory mechanisms. The interaction of four cAMP molecules with the R-subunits leads to the dissociation of the protein kinase complex, releasing the C-subunits, which are then capable of catalyzing the corresponding reaction (i.e., phosphorylating target cellular proteins):

An activated protein kinase can phosphorylate several hundred or thousands of substrate proteins, leading to a significant Amplification of the initial chemical regulatory signal—a cascade regulation system. The molecular mechanisms underlying the activation of cAMP-dependent biochemical reaction cascades in response to hormonal action on sensitive cells will be examined in greater detail in Chapter 23.

Impairment of Enzymatic Processes

A decrease in the concentration of any enzyme within a biochemical system in vivo results in the disruption of the corresponding enzymatic reaction. Such disruptions of enzymatic processes occur in the human body and form the biochemical basis for The Development of numerous pathological conditions. When a reduction in the amount of enzyme along a specific metabolic pathway stems from a cell's congenital inability to biosynthesize it due to hereditary genetic factors, it leads to inborn errors of metabolism.

THE CONCEPT OF "inborn errors of metabolism" was proposed back in 1908 by the physician A. Garrod, who was the first to recognize the hereditary nature of such Metabolic Disorders in humans as alkaptonuria, cystinuria, albinism, and pentosuria. Thanks to the development of biochemical genetics, it has been established that the Molecular Basis of inborn errors of metabolism lies in enzyme defects (enzymopathies) caused by Mutations in the genes responsible for the Synthesis of specific enzyme proteins.

Enzymopathies include:

- inborn errors of simple and complex Carbohydrate Metabolism;

- inborn errors of lipid metabolism;

- inborn errors of Amino acid metabolism;

- inborn errors of porphyrin metabolism;

- inborn errors of purine and pyrimidine metabolism.

To date, about 150 hereditary enzymopathies are known. Naturally, if the Gene whose expression proceeds via the synthesis of an enzyme protein is dominant, the corresponding metabolic pathology manifests even in the heterozygous state; in the case of a defect in The structure of a recessive gene, individuals heterozygous for this gene are still able to synthesize the respective protein in reduced amounts, and the enzymopathy as a hereditary pathology often manifests itself only under specific physiological conditions.

The biochemical mechanisms underlying specific types of enzymopathies and their clinical manifestations will be discussed in the relevant chapters.