Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Enzymes
Regulation of Enzyme Activity

One of the unique properties of living organisms is their remarkable ability to maintain a balance between catabolic (biodegradative) and anabolic (biosynthetic) processes. At the same time, Cells simultaneously carry out the synthesis, breakdown, and interconversion of hundreds and thousands of diverse substances, which in turn are regulated by numerous mechanisms ensuring the constancy of the internal environment of the Organism. Some of these regulatory mechanisms, among which an important role belongs to the mechanisms regulating the synthesis and catalytic activity of Enzymes, will be discussed below.

The Influence of the law of mass action. In an enzyme-catalyzed reversible chemical reaction, such as A + B <=> C + D, the concentration of the reaction components and, accordingly, the direction of the reaction will be regulated by the law of mass action. This can be demonstrated, in particular, in the reversible Transamination reaction catalyzed by the enzyme Alanine aminotransferase:

Class="center">Alanine + a-Ketoglutarate <=> Pyruvate + Glutamate.

This type of regulation obviously plays only a limited role, since under real conditions the reaction usually proceeds in one direction, as the resulting products may serve as substrates for the action of Other Enzymes and be removed from the reaction sphere. In these cases, a steady state is established rather than true equilibrium.

Changes in Enzyme Concentration. The phenomenon of induced (inducible) enzyme synthesis has been well studied in Bacteria when grown on a medium where a particular carbohydrate, such as glucose, serves as the sole source of carbon and energy. Replacing glucose with lactose (the inducer) in the medium leads to the induced or adaptive (after a short lag phase) Synthesis of the enzyme galactosidase (programmed by the lactose Gene, see Chapter 13), which cleaves lactose into glucose and galactose.

Prokaryotic and Eukaryotic cells contain enzymes whose concentration does not require The addition of an inducer; these are the so-called constitutive enzymes. The amount of an enzyme in a Cell depends on the presence of the product of the reaction catalyzed by that enzyme, with the reaction product inhibiting enzyme synthesis As a result of repression (see below).

In animal Tissues, rapid enzyme synthesis is observed less frequently. Its mechanism (inducible synthesis) has been studied for only a small number of enzymes—Tyrosine transaminase, Serine and Threonine dehydratase, Tryptophan pyrrolase, etc.—in response to hormone administration or protein ingestion. However, upon exposure of the organism to certain poisons, carcinogenic substances, Alkaloids, or insecticides, a sharp increase in the activity (and accordingly, the amount) of Endoplasmic reticulum hydroxylase (monooxygenase) enzymes in Liver cells is observed after a few days; these enzymes oxidize foreign substances into products that are non-toxic to the organism. It is quite reasonable to assume that in these cases enzyme synthesis occurs via induction (i.e., de novo). Cases have been described where, under the action of such hydroxylases, foreign substances are converted in the body into more toxic compounds. This phenomenon, the reverse of detoxification, is termed Lethal synthesis.

Proenzymes. Proteolytic Enzymes of the digestive tract, as well as the Pancreas, are synthesized in an inactive form as proenzymes (zymogens). Regulation in these cases comes down to The conversion of proenzymes into active enzymes under METABOLISM/18.html">The Influence of specific agents or other enzymes—proteinases. Thus, Trypsin is synthesized in The Pancreas in the form of inactive trypsinogen. Upon entering the intestine, it is converted into active trypsin as a result of autocatalysis or under the action of other proteinases (the activation mechanism is discussed in detail in Chapter 12). The conversion of inactive pepsinogen into active Pepsin occurs autocatalytically as a result of specific Limited proteolysis in the presence of Hydrochloric acid and is also associated with the Cleavage of a peptide-type specific inhibitor from the proenzyme. These conversions of zymogens into active enzymes are associated with Conformational Changes in the enzyme molecule and The formation of the Active Site or its exposure (unmasking). The synthesis of proteinases and A number of other inactive precursor Proteins in an inactive form evidently has a definite biological meaning, preventing the destruction of the Cells of the Organs in which the proenzymes are produced. Examples of such protein activation include the activation of certain Hormones (proinsulin → Insulin), Connective Tissue protein (soluble procollagen is converted into insoluble Collagen), and Blood clotting proteins.

Fig. 4.23. Covalent Modification of an enzyme by phosphorylation-dephosphorylation of serine residues.

Fig. 4.24. Non-covalent modification of an enzyme by adenylylation-deadenylylation.

Chemical modification of enzymes. During the formation of their tertiary Structure, some proteins undergo post-synthetic chemical modification (see Chapter 1). It has been found that The activity of a number of Key Enzymes in Carbohydrate Metabolism, in particular phosphorylase, Glycogen synthase, etc., is also controlled by phosphorylation and dephosphorylation carried out by specific enzymes—protein kinase and protein phosphatase—whose activity in turn is regulated by hormones (see Chapter 10). The level of activity of key carbohydrate metabolism enzymes and, consequently, the rate and direction of metabolic processes themselves are determined by The ratio of the phosphorylated and dephosphorylated forms of these enzymes.

A distinction is usually made between reversible covalent and non-covalent chemical modifications of enzymes, carried out via the OH groups of serine, less frequently of tyrosine, or through non-covalent interactions with the enzyme molecule. In the first case, the active enzyme is either the phosphorylated or dephosphorylated form, as in the case of Muscle phosphorylase and glycogen synthase molecules, respectively (see Chapter 10). As examples, both types of modification can be represented schematically, where the symbol P denotes a phosphate residue, P_i is inorganic phosphate (H₃PO₄), PP_i is inorganic pyrophosphate (H₄P₂O₇), and AMP is an adenylic acid residue (Figs. 4.23 and 4.24).

Chemical post-synthetic modification of enzymes also includes processes of limited proteolysis (see above), methylation (see Chapter 13), glycosylation, uridylylation, adenylylation, ADP-ribosylation*, etc., thereby providing a microscopic type of Introduction/15.html">Regulation of enzyme Activity and, accordingly, the physiological rate of metabolic processes.

* Interestingly, diphtheria and cholera toxins possess enzymatic activity, causing ADP-ribosylation (and consequent inactivation) of key cellular enzymes or proteins. Diphtheria toxin turns off the synthesis of protein factor 2 of the elongation stage of Protein Synthesis, while cholera toxin turns off a specific G-protein, resulting in massive Water loss.

Allosteric Regulation. In many strictly biosynthetic reactions, the main type of Regulation of the rate of a multi-step enzymatic process is feedback inhibition. This means that the end product of the biosynthetic chain inhibits the activity of The enzyme catalyzing The First stage of synthesis, which is key for this reaction chain. Since the end product is structurally different from the substrate, it binds to the allosteric (non-catalytic) site of the enzyme molecule, causing inhibition of the entire synthetic reaction chain.

Suppose that a multi-step biosynthetic process takes place in cells, each stage of which is catalyzed by its own enzyme:

The rate of such a total sequence of reactions is largely determined by the concentration of the end product P, the accumulation of which above the permissible level exerts a powerful inhibitory effect on the first stage of the process and, accordingly, on the enzyme E₁.

The existence of such a mechanism for controlling enzyme activity by metabolites was first discovered in E. coli during studies on the synthesis of isoleucine and CTP. It turned out that isoleucine, the end product of synthesis, selectively suppresses the activity of threonine dehydratase, which catalyzes the first stage of the sequential process of converting threonine into isoleucine, comprising five enzymatic reactions:

Similarly, CTP as the end product of the biosynthetic pathway exerts an inhibitory effect on the first enzyme (aspartate carbamoyltransferase), thereby regulating its own synthesis (see Chapter 13). This type of inhibition is termed feedback inhibition, or retroinhibition. Its existence has been proven in All living organisms. Currently, it is considered one of the leading types of regulation of enzyme activity and cellular Metabolism as a whole*.

On the other hand, in amphibolic processes that simultaneously perform biosynthetic and biodegradative Functions**, the existence of regulation both by retroinhibition and by high-energy compounds—indicators of the cellular energy state—has been proven. For amphibolic processes, a unique type of regulation intrinsic only to them is, in addition, activation by a precursor, when the first metabolite in a multi-step pathway activates the enzyme catalyzing the final step. Thus, the activating effect of glucose-6-phosphate, which is a precursor of glycogen, on the enzyme glycogen synthase has been proven.

* The reaction rate (as well as enzyme activity) in purely biodegradative (catabolic) processes is regulated by intermediates that serve as indicators of the cellular energy state (purine NUCLEOTIDES, pyrophosphate, inorganic phosphate, etc.).

** Amphibolic processes include such central metabolic pathways as Glycolysis, Glycogenolysis, The Tricarboxylic Acid Cycle, the Hexose monophosphate pathway, and AMINO ACID TRANSAMINATION.

Fig. 4.25. Interaction of an allosteric enzyme with a substrate and effectors (diagram). a — active complex; b — inactive complex; 1 — active site; 2 — allosteric site; 3 — substrate; 4 — positive effector; 5 — negative effector.

Such types of end-product inhibition and first-product activation are characteristic of allosteric (regulatory) enzymes, in which an effector, or modulator—structurally distinct from the substrate—binds to a specific (allosteric) site on the enzyme molecule that is spatially remote from the active site. It should be borne in mind, however, that modulators of allosteric enzymes can act as either activators or inhibitors. Frequently, the substrate itself exerts an activating effect. Enzymes for which both the substrate and the modulator share identical structures are termed homotropic, in contrast to heterotropic enzymes, in which the modulator has a different structure from the substrate. The interconversion of active and inactive allosteric forms, along with the conformational changes observed upon the binding of the substrate and effectors, is illustrated in Fig. 4.25. The binding of a negative effector to the allosteric site induces significant alterations in the configuration of the enzyme molecule's active site, causing the enzyme to lose its affinity for the substrate (formation of an inactive complex).

Allosteric interactions are manifested in the shape of the curves representing the dependence of the initial reaction rate on substrate or effector concentration, specifically in the sigmoidal (S-shaped) nature of these curves (deviating from the hyperbolic Michaelis-Menten curve). The sigmoidal dependence of v on [S] in the presence of a modulator is due to the cooperativity effect. This means that the binding of one substrate molecule facilitates the binding of the next molecule at the active site, thereby promoting an increase in reaction rate. Furthermore, allosteric regulatory enzymes are characterized by a non-linear dependence of reaction rate on Substrate Concentration.

Other types of ENZYME ACTIVITY REGULATION. The absolute amount of an enzyme present in a cell is regulated by The kinetics of its Synthesis and degradation. Regulatory mechanisms also include competition between enzymes for a common substrate, the suppression of activity of one of the Isoenzymes (in multiple enzyme forms), the influence of cofactor concentrations, and the phenomenon of compartmentalization. The Mechanism of metabolic compartmentalization presumably plays a vital biological role by spatially separating enzymes from their substrates via Biomembranes (for example, isolating lysosomal enzymes—such as proteinases, Phosphatases, ribonucleases, and other hydrolytic enzymes—from the cytoplasmic components upon which they act). Furthermore, by facilitating independent regulation, this mechanism allows The Cell to segregate metabolic processes that are incompatible when occurring in the same Location (and potentially at the same time). An example of the latter is The pathway of higher fatty acid synthesis, which predominantly occurs in the soluble cytoplasmic fraction, and the pathway of fatty acid degradation (oxidation), which is localized within Mitochondria. It must be noted, however, that compartmentalization creates The Challenge of transporting both metabolites and reducing equivalents across the biomembranes of subcellular Organelles. This task is resolved by the so-called shuttle mechanism, which converts metabolites into forms capable of crossing membranes, thereby maintaining intracellular Homeostasis (see Chapter 13).