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

Enzymes
Basic Properties of Enzymes
Enzyme Activation and Inhibition

The rate of an enzymatic reaction, much like enzyme activity, is largely determined by the presence of activators and inhibitors in the medium: the former accelerate the reaction, while the latter inhibit it. An activating effect on the rate of an enzymatic reaction can be exerted by A wide variety of organic and inorganic substances. For instance, Hydrochloric acid activates the action of Pepsin in gastric juice;

Bile acids enhance The activity of pancreatic lipase; certain tissue Enzymes (oxidoreductases, cathepsins, arginase), plant proteinase, etc., are significantly activated by compounds containing free SH-groups (Glutathione, Cysteine), and A number of enzymes are also activated by Vitamin C. Divalent and, less frequently, monovalent Metal Ions most commonly act as activators. Evidence has been obtained that approximately a quarter of all known enzymes require the presence of metals to exhibit full catalytic activity. Many enzymes are completely inactive in the absence of metals. For instance, upon the removal of zinc, Carbonic anhydrase—which catalyzes the Biosynthesis and breakdown of H₂СО₃—practically loses its enzymatic activity; moreover, zinc cannot be replaced by any other metal in this regard. Some enzymes* are known whose action is activated by ions of several metals; in particular, enolase is activated by Mg²⁺, Mn²⁺, and K⁺ (Table 4.4).

Class="center">Table 4.4. Metal-Activated Enzymes

The Molecular Mechanism of metal action in Enzymatic Catalysis, or The Role of metals in enzyme activation. In a number of cases, metal ions (Co²⁺, Mg²⁺, Zn²⁺, Fe²⁺) function as prosthetic groups of enzymes, serve as electron acceptors and Donors, or act as electrophiles or nucleophiles, maintaining reactive groups in the proper orientation. In other instances, they facilitate the attachment of the substrate to the Active Site and The formation of the enzyme-substrate complex. For example, Mg²⁺ ions, via the negatively charged phosphate group, ensure the binding of monophosphate esters of organic substances to the Active Site of phosphatases, which catalyze the Hydrolysis of these compounds. Sometimes the metal binds to the substrate to form a true substrate upon which the enzyme acts. Specifically, Mg²⁺ ions activate creatine phosphokinase through the formation of a true substrate—the magnesium salt of ATP. Finally, there is experimental Evidence of the direct participation of metals (e.g., Ca²⁺ ions in the salivary amylase molecule) in the formation and stabilization of the active site and the overall three-dimensional Structure OF THE enzyme molecule. It should also be noted that metals frequently act as allosteric modulators (effectors; see Fig. 4.22). By interacting with the allosteric center, such a metal (effector) promotes the Formation of the most favorable spatial conformation of the enzyme and the active enzyme-substrate complex.

* It is usually difficult to draw a clear line between metalloenzymes (where the metal is firmly bound to the protein and indispensable) and metal-activated enzymes (the latter merely accelerate the reaction and dissociate easily).

Anions at physiological concentrations are generally ineffective or exert a minor activating effect on enzymes. Exceptions include pepsin, certain oxidoreductases activated by anions, as well as salivary amylase (which catalyzes starch hydrolysis), whose activity increases under the action of chloride ions, and adenylate cyclase, which is activated by halide anions.

Enzyme Inhibitors are generally divided into two broad classes: reversible and irreversible. These are substances that cause partial (reversible) or complete inhibition of enzyme-catalyzed reactions. Antienzymes (also known as antienzymes or antizymes) have recently been discovered; these are Proteins (or Polypeptides) that act as enzyme inhibitors. Such substances include, for example, the Trypsin inhibitor found in soybeans and serum antitrypsin. The antienzyme of Ornithine decarboxylase was recently discovered in animal Liver (see Chapter 12). Antizymes most likely form poorly dissociable complexes with their respective enzymes, knocking them out of Chemical Reactions. Sometimes an inhibitor is an integral component of an enzyme precursor, such as pepsin (see Chapter 12), or is part of complex enzyme complexes, such as protein kinase and protein phosphatase, which catalyze phosphorylation-dephosphorylation processes in living organisms. However, it remains unclear whether such antienzymes are true inhibitors or Regulatory Subunits, and in particular, what the difference is in the function of the regulatory (R) subunit within protein kinase and the inhibitory (I) subunit within protein phosphatase.

Because enzymes are proteins, any agents that cause Protein Denaturation (acids, alkalis, heavy metal salts, heating) lead to irreversible Enzyme inactivation. However, such inactivation is relatively nonspecific and is not related to the METABOLISM/10.html">Mechanism of enzyme Action. A much larger group consists of so-called specific inhibitors, which exert their effect on a single enzyme or a group of related enzymes, causing reversible or irreversible inhibition. The Study of these inhibitors is of great importance. First, inhibitors can provide valuable information about the Chemical Nature of an enzyme's active site, as well as the composition of its functional groups and The Nature of the chemical bonds that ensure the formation of the enzyme-substrate complex. Substances are known, including pharmaceutical drugs, that specifically bind to a particular functional group in the enzyme molecule, rendering it out of the chemical reaction. For instance, iodoacetate ICH₂—COOH, its amide and ethyl ester, para-chloromercuribenzoate ClHg—C₆H₄—COOH, and Other Reagents react relatively easily with certain SH-groups of enzymes. If such groups are essential for the catalytic act, The addition of these inhibitors leads to a complete loss of enzyme activity:

R-SH + ICH-СООН → HI + R-S-CH₂-COOH

The action of a number of Other Enzymes (cholinesterase, trypsin, and Chymotrypsin) is strongly inhibited by certain organophosphorus compounds, such as DFP, due to the blockage of the key Serine hydroxyl group at the active site (see above).

Second, inhibitors have found widespread use in enzymology for investigating the nature of multiple enzyme forms and Isoenzymes, which differ not so much in their electrophoretic mobility as in their varying sensitivity to the same inhibitor.

With the aid of inhibitors that block individual stages of a multi-step metabolic process, researchers can precisely determine not only The sequence of chemical reactions but also the Nature of the enzymes participating in these transformations. By employing this approach with iodoacetate, fluorides, and other specific inhibitors, The Glycolytic Pathway of the oxidation-reduction transformations of glucose up to the stage of lactic acid formation in Muscle tissue—comprising 11 steps involving 11 enzymes and 10 intermediate metabolites—was successfully deciphered.

The MECHANISM OF ACTION of many toxins and poisons on the Organism is associated with Enzyme Inhibition. It is known that in cases of poisoning by hydrocyanic acid salts, death occurs As a result of the complete inhibition and shut down of tissue respiratory enzymes (the cytochrome system), particularly in Brain Cells. The Toxic Effect of certain insecticides on humans and animals is caused by the inhibition of cholinesterase activity—an enzyme that plays a key role in The Nervous system.

Modern, so-called rational Chemotherapy (the targeted use of medicinal drugs in medicine) must be based on a precise understanding of The Mechanism of action of drugs on enzyme biosynthesis, on the activity of already synthesized enzymes, or on The regulation of their activity in the body. Selectively acting inhibitors are sometimes used to treat certain diseases. For instance, trasylol, an inhibitor of a number of proteinases (trypsin, chymotrypsin, and kallikrein), is widely used to treat acute pancreatitis—a condition in which Blood levels of trypsin and chymotrypsin rise sharply. An understanding of the selective inhibitory Introduction/43.html">Action of Certain natural and synthetic compounds (so-called antimetabolites) on enzymes can serve as a methodological foundation for developing effective Methods of synthesizing chemotherapeutic drugs. This pathway opens up broad opportunities for targeted intervention in enzyme synthesis within the body and for regulating metabolic intensity in pathology.

Types of inhibition. Reversible and irreversible inhibition are distinguished. If an inhibitor causes persistent Changes in the spatial Tertiary Structure of the enzyme molecule or Modification of the enzyme's functional groups, this type of inhibition is termed irreversible. More frequently, however, reversible inhibition occurs, which is amenable to quantitative analysis based on the Michaelis-Menten Equation. Reversible inhibition is, in turn, subdivided into competitive and noncompetitive, depending on whether or not the inhibition of the enzymatic reaction can be overcome by increasing the Substrate Concentration.

Competitive inhibition can be caused by substances whose structure resembles that of the substrate, yet differs slightly from The structure of the true substrate. This type of inhibition is based on the binding of the inhibitor to the substrate-binding (active) site. A classic example of this type of inhibition is the inhibition of succinate dehydrogenase (SDH) by malonic acid. This enzyme catalyzes the oxidation via dehydrogenation of succinic acid (succinate) into fumaric acid:

If malonate (an inhibitor) is added to the medium, its structural similarity to the true substrate, succinate (the presence of two identical ionized carboxyl groups), causes it to interact with the active site, forming an enzyme-inhibitor complex; however, The transfer of a hydrogen atom from malonate is completely precluded. The structures of the substrate (succinate) and inhibitor (malonate) still differ slightly. Therefore, they compete for binding to the active site, and the degree of inhibition will be determined by The ratio of malonate to succinate concentrations rather than the absolute concentration of the inhibitor. Thus, the inhibitor can reversibly bind to the enzyme, forming an enzyme-inhibitor complex. This type of inhibition is sometimes referred to as metabolic antagonism-type inhibition (Fig. 4.20).

In general terms, the reaction of an inhibitor interacting with an enzyme can be represented by the following equation:

The resulting complex, termed the enzyme-inhibitor complex EI, unlike the enzyme-substrate complex ES, does not break down to yield reaction products. Following the Michaelis-Menten theory, the dissociation constant of the EI complex, or the inhibition constant K_i, can be defined as the ratio of the reverse and forward reaction rate constants:

that is, the inhibition constant is directly proportional to the product of the enzyme and inhibitor concentrations and inversely proportional to the concentration of the EI complex.

The method of competitive inhibition has found widespread application in medical practice. It is known, for instance, that sulfonamide drugs are used to treat certain bacterial infectious diseases. It turned out that these drugs bear a structural resemblance to Para-aminobenzoic Acid, which bacterial cells utilize to synthesize Folic acid, an essential component of bacterial enzymes. Owing to this structural similarity, sulfonamides block enzyme action by displacing para-aminobenzoic acid from its complex with the enzyme that synthesizes folic acid, ultimately leading to the inhibition of bacterial growth.

Fig. 4.20. Action of a competitive inhibitor (diagram based on V.L. Kretovich).

E - enzyme; S - substrate; P₁ and P₂ - reaction products; I - inhibitor.

Some analogues of vitamin B₆ and folic acid, notably deoxypyridoxine and aminopterin (see Chapter 7), act as competitive, so-called coenzyme inhibitors (or Antivitamins), which inhibit numerous biological processes running intensively under pathological conditions. The Use of such analogues in medical practice (particularly in dermatology and oncology) is based on the competitive displacement of Coenzymes from the substrate-binding sites of key metabolic enzymes.

Non-competitive inhibition is caused by substances that lack structural similarity to substrates and often bind not to the active site, but to another region of the enzyme molecule. The degree of inhibition in many cases is determined by the duration of the inhibitor's action on the enzyme. With this type of inhibition, due to the formation of a stable covalent bond, the enzyme often undergoes complete inactivation, and then the inhibition becomes irreversible. Examples of irreversible inhibition include the action of iodoacetate, DFP, as well as diethyl-n-nitrophenyl phosphate and cyanide salts. This action consists in the binding and blocking of functional groups or metal ions in the enzyme molecule.

It should be noted that non-competitive inhibition can also be both reversible and irreversible, since there is no competition between the substrate and the inhibitor for the active site. Examples of irreversible inhibition were given earlier. In reversible non-competitive inhibition, the substrate S and the inhibitor I bind to different sites, thus making it possible to form both the EI complex and the ternary EIS complex; the latter can dissociate to release the product, but at a lower rate than the ES complex.

This type of non-competitive inhibition is most frequently observed in enzymes that catalyze The conversion of more than one substrate, when inhibitor binding does not block the binding of the substrate to the active site. In this case, the inhibitor binds to both the free enzyme and the ES complex.

Furthermore, so-called uncompetitive inhibition is known, in which the inhibitor also binds to the enzyme at a non-catalytic site, but not to the free enzyme, and only to the ES complex in the form of a ternary complex.

To resolve the question of the type of inhibition, the Michaelis-Menten, Lineweaver-Burk equations, or others—such as the Eadie-Hofstee equation—are used:

along with the corresponding plots in rectilinear coordinates.

In competitive inhibition, the inhibitor increases the K_m value without affecting the maximum velocity V_max (Fig. 4.21). This means that at a sufficiently high substrate concentration [S], the inhibitor is displaced from the EI complex by substrate molecules. In non-competitive inhibition (Fig. 4.22), the inhibitor decreases the maximum velocity. If the K_m value does not decrease in this process, it is referred to as pure non-competitive inhibition. This type of inhibition occurs upon the formation of inactive, poorly dissociating a - in coordinates of v versus [S]; b - in coordinates of 1/v versus 1/[S]; V_max and V_i - maximum reaction velocities; K_m and K_mi - Michaelis constants in the absence (1) and presence (2) of the inhibitor, respectively.

Fig. 4.21. Plots of enzymatic reaction rate versus substrate concentration in the presence of a competitive inhibitor.

Fig. 4.22. Plots of enzymatic reaction rate versus substrate concentration in the presence of a non-competitive inhibitor. Designations are the same as in Fig. 4.21.

EI and (or) EIS complexes. Frequently, however, a mixed type of inhibition is observed—sometimes called partially non-competitive or reversible non-competitive inhibition (see above)—in which a decrease in V_max is combined with a simultaneous increase in K_m values. This means that the EI complex retains partial activity, i.e., The ability to form an intermediate ternary EIS complex in which the substrate undergoes delayed catalytic conversion. In rare cases, the degree of inhibition of enzyme activity may increase with increasing substrate concentration. For this type of inhibition, the rather inaccurate term "uncompetitive inhibition" has been proposed, as noted earlier. One of the mechanisms for such inhibition is due to the possibility of the inhibitor binding to the ES complex to form an inactive or slowly reacting ternary EIS complex.

Thus, graphical analysis of enzymatic reaction rates as a function of substrate concentrations can yield valuable information not only on the Kinetics of Enzymatic reactions, but also on the molecular Mechanisms of Enzymatic Catalysis.