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

SECTION 2. ENZYMOLOGY

VI. Inhibition of Enzyme Activity

The term "Enzyme Inhibition" refers to the reduction of catalytic activity in the presence of specific substances known as inhibitors. Inhibitors are defined as agents that cause a decrease in enzyme activity. It should be noted that all Denaturing Agents also reduce The rate of any enzymatic reaction due to the nonspecific Denaturation of the protein molecule; therefore, denaturing agents are not classified as true inhibitors.

Inhibitors are of great interest for elucidating the Mechanisms of Enzymatic Catalysis and helping to determine The Role of individual Enzymes in metabolic pathways. The action of many medicinal drugs and poisons is based on the inhibition of enzyme activity; consequently, understanding the mechanisms of this process is crucial for molecular pharmacology and toxicology.

Inhibitors can interact with enzymes with varying degrees of affinity, forming the basis for the distinction between reversible and irreversible inhibition. Based on their MECHANISM OF ACTION, inhibitors are subdivided into Competitive and non-competitive.

A. Reversible Inhibition

Reversible inhibitors bind to the enzyme via weak non-covalent bonds and can easily dissociate from the enzyme under certain conditions. Reversible inhibitors are categorized into competitive and non-competitive.

1. Competitive Inhibition

Competitive inhibition refers to the reversible decrease in the rate of an enzymatic reaction caused by an inhibitor that binds to the Active Site of the enzyme, thereby preventing The formation of the enzyme-substrate complex. This type of inhibition is observed when the inhibitor is a structural analog of the substrate, leading to competition between substrate and inhibitor molecules for the binding site within the enzyme's active site. In this case, either the substrate or the inhibitor interacts with the enzyme, forming either enzyme-substrate (ES) or enzyme-inhibitor (EI) complexes. When the enzyme-inhibitor complex (EI) is formed, no reaction product is generated (Fig. 2-21).

Class="center">Fig. 2-21. Scheme of competitive enzyme inhibition.

The following equations apply to competitive inhibition:

Е + S <=> ЕS —> Е + Р,

Е + I <=> ЕI.

A classic example of competitive inhibition is the inhibition of the succinate dehydrogenase reaction by malonic acid (Fig. 2-22). Malonic acid is a structural analog of succinate (possessing two carboxyl groups) and can similarly interact with the active site of succinate dehydrogenase. However, the removal of two hydrogen atoms from malonic acid is impossible; consequently, the reaction rate decreases.

Fig. 2-22. Example of competitive inhibition of succinate dehydrogenase by malonic acid. I - succinate binds to the active site of the enzyme succinate dehydrogenase; II - during the enzymatic reaction, two hydrogen atoms are removed from succinate and transferred to the coenzyme FAD, resulting in the formation of fumarate, which is released from the active site of succinate dehydrogenase; III - malonic acid, a structural analog of succinate, also binds to the active site of succinate dehydrogenase, but no chemical reaction takes place.

Kinetic Parameters

Competitive Inhibitors reduce the rate of a chemical reaction. A competitive inhibitor increases the K_m for a given substrate (decreasing the substrate's affinity for the enzyme). This means that in the presence of a competitive inhibitor, a higher Substrate Concentration is required to achieve 1/2 V_max.

Increasing The ratio of substrate to inhibitor concentration diminishes the degree of inhibition. At significantly higher substrate concentrations, inhibition is completely overcome because the active sites of all enzyme molecules will predominantly exist in the complex with the substrate.

Medicinal Drugs as Competitive Inhibitors

Many drugs exert their therapeutic effects through a mechanism of competitive inhibition. For example, quaternary ammonium compounds inhibit acetylcholinesterase, which catalyzes the Hydrolysis of acetylcholine into Choline and acetic acid (see the scheme below).

Upon The addition of inhibitors, acetylcholinesterase activity decreases while the concentration of acetylcholine (the substrate) increases, which is accompanied by enhanced Nerve Impulse transmission. Cholinesterase inhibitors are used in the Treatment of muscular dystrophies. Effective anticholinesterase drugs include prostigmine (neostigmine), edrophonium, and others (Fig. 2-23).

Fig. 2-23. Diagram of the active site of acetylcholinesterase. A - binding of acetylcholine at the active site of the enzyme. The arrow indicates the site of ester bond hydrolysis in the acetylcholine molecule; B - binding of the competitive inhibitor proserine at the active site of the enzyme. The site of proserine hydrolysis is indicated, although the reaction proceeds much slower than with acetylcholine; C - binding of the competitive inhibitor edrophonium at the active site of the enzyme. Edrophonium binds to the active site of acetylcholinesterase, preventing the binding of acetylcholine.

Antimetabolites as Medicinal Drugs

Substances known as antimetabolites are used in medical practice as competitive Enzyme Inhibitors. Being structural analogs of natural substrates, these compounds cause competitive enzyme inhibition on the one hand, and on the other hand, they can be utilized by the same enzymes as pseudosubstrates, leading to the synthesis of anomalous products. Anomalous products lack functional activity; As a result, a decrease in the rate of certain metabolic pathways is observed.

The following antimetabolites are used as medicinal drugs: sulfonamide drugs (analogs of Para-aminobenzoic Acid) used for the treatment of infectious diseases (see Chapter 9), and nucleotide analogs for the treatment of oncological diseases (see Chapter 10).

2. Noncompetitive Inhibition

Noncompetitive inhibition of an enzymatic reaction is defined as inhibition in which the inhibitor interacts with the enzyme at a site other than the active site (Fig. 2-24). Noncompetitive inhibitors are not structural analogs of the substrate.

A noncompetitive inhibitor can bind to either the free enzyme or the enzyme-substrate complex, forming an inactive complex. The binding of a noncompetitive inhibitor induces a conformational change in the enzyme molecule such that the interaction of the substrate with the active site is disrupted, leading to a decrease in the rate of the enzymatic reaction.

Fig. 2-24. Diagram of noncompetitive inhibition of enzyme activity.

Kinetic Dependencies

The kinetic dependence of Noncompetitive inhibition is presented in Fig. 2-25. This type of inhibition is characterized by a decrease in the Vm_ах of the enzymatic reaction and a reduction in the affinity of the substrate for the enzyme, i.e., an increase in K_m.

Fig. 2-25. Effect of a noncompetitive inhibitor on the enzymatic reaction rate as a function of substrate concentration. V_mах is the maximum reaction rate in the absence of the inhibitor; '\/_mах is the maximum reaction rate in the presence of the inhibitor; K_m is the Michaelis constant in the absence of the inhibitor; 'К_m is the Michaelis constant in the presence of the inhibitor.

B. Irreversible Inhibition

Irreversible inhibition is observed when stable covalent bonds are formed between the inhibitor and enzyme molecules. Most frequently, the active site of the enzyme undergoes modification. As a result, the enzyme loses its ability to perform its catalytic function.

Irreversible inhibitors include heavy Metal Ions such as mercury (Нg²⁺), silver (Аg⁺), and arsenic (Аs³⁺), which at low concentrations block The sulfhydryl groups of the active site. In this case, the substrate cannot undergo chemical transformation (Fig. 2-26). In the presence of reactivators, the Enzymatic Function is restored. At high concentrations, heavy metal ions cause denaturation of the protein molecule of the enzyme, leading to its complete inactivation.

Fig. 2-26. Mechanism of action of mercury ions as an irreversible inhibitor. Mercury ions at low concentrations block the sulfhydryl groups of the active site, leading to a decrease in the rate of the enzymatic reaction.

1. Specific and Nonspecific Inhibitors

The Use of irreversible inhibitors is of great interest for elucidating the METABOLISM/10.html">Mechanism of enzyme Action. For this purpose, substances that block specific groups at the active site of enzymes are employed. Such inhibitors are termed specific. A number of compounds readily react with specific chemical groups. If these groups participate in catalysis, complete inactivation of the enzyme occurs.

The role of Serine hydroxyl groups in the catalytic mechanism is investigated using fluorophosphates, such as diisopropyl fluorophosphate. Diisopropyl fluorophosphate (DFP) specifically reacts with only one of the many serine residues in the active site of the enzyme. The Ser residue capable of reacting with DFP has an identical or very similar amino acid environment (Table 2-2). The high reactivity of this residue compared to other Ser residues is due to amino acid residues that are also part of the active site of the enzymes.

Table 2-2. Investigation of The sequence of amino acid residues surrounding the reactive serine residue interacting with DFP

DFP is classified as a specific irreversible inhibitor of "serine" enzymes because it forms a covalent bond with the hydroxyl group of serine located in the active site and plays a key role in the catalytic process (Fig. 2-27).

Fig. 2-27. Inhibition of chymotrypsin activity by diisopropyl fluorophosphate.

Iodoacetate and p-chloromercuribenzoate readily react with the SH groups of protein Cysteine residues (Fig. 2-28). Because these inhibitors react with any free protein SH groups, they are classified as non-specific rather than specific inhibitors. If SH groups are directly involved in catalysis, these inhibitors can be used to determine the role of enzyme SH groups in the catalytic process.

Рис. 2-28. Ингибирование активности ферментов вследствие ковалентной модификации остатков цистеина.

2. Irreversible enzyme inhibitors as drugs

An example of a drug whose mechanism of action relies on irreversible enzyme inhibition is the widely used medication aspirin. The nonsteroidal anti-inflammatory drug aspirin exerts its pharmacological effect by inhibiting cyclooxygenase, the enzyme that catalyzes the formation of Prostaglandins from arachidonic acid. Through a chemical reaction, the acetyl group of aspirin attaches to the free terminal NH₂ group of one of the cyclooxygenase subunits (see diagram below).

This leads to a reduction in The production of prostaglandin reaction products (see Section 8), which serve a wide range of biological Functions, including acting as mediators of inflammation.