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

Chapter II. GENERAL PRINCIPLES OF METABOLISM

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

7.1. MECHANISMS OF ENZYME ACTION

Enzymes increase the rates of the biochemical reactions they catalyze by 10⁸-10²⁰-fold; in the absence of an enzyme, virtually any metabolic reaction fails to proceed. It is known that the rate constant of a chemical reaction depends on its activation energy and Temperature, as expressed by the Arrhenius equation in exponential form:

Class="center">k = Ae^-ΔE/RT.

In chemical Thermodynamics, activation energy (ΔE in the Arrhenius equation) refers to the additional energy required for molecules (substrates S) to transition into the transition (activated) state (S*), which precedes their conversion into reaction products. Accordingly, the exponential term of the equation e^-ΔE/RT (the Boltzmann factor) represents the fraction of molecules in the system that possess sufficient energy for chemical transformation.

Since all metabolic processes in living organisms occur under isothermal conditions, the catalytic action of enzymes is realized through the lowering of the activation energy (ΔE) of the biochemical reaction by the enzymes themselves, which increases the Boltzmann factor and, consequently, the reaction rate constant by several orders of magnitude:

Fig. 7.1. Energy diagrams of a chemical reaction (S → P) without a catalyst (1) and in the presence of an enzyme catalyst (2). ΔE and ΔE_k are the activation energies of the reaction without and in the presence of the catalyst, respectively.

Compared to chemical catalysts, enzymes significantly reduce the ΔE of the reactions they catalyze. For example, in the hydrogen peroxide decomposition reaction

the activation energy can be reduced under the action of inorganic catalysts (iodide, platinum) or, to a much greater extent, under the Influence of the specific enzyme catalase, which leads to corresponding increases in the reaction rate.

Table 7.1. Non-enzymatic and Enzymatic Catalysis of hydrogen peroxide decomposition (t = 25 °C; the rate constant of the uncatalyzed reaction is taken as unity) (according to S. Rapoport, 1964)

Active Sites of Enzymes

The decrease in the activation energy of a biochemical reaction and the resulting high catalytic efficiency of enzymes are achieved through the interaction of substrates with specific Regions of the enzyme molecule (active or catalytic sites), which is accompanied by the close approximation and orientation of the corresponding chemical groups of the substrates, creating the steric conditions necessary for specific catalytic acts to take place.

The Active Site is a region of the enzyme protein molecule that interacts with the substrate during an enzymatic reaction and is essential for substrate conversion in the catalytic process.

It is formed by specific segments of the polypeptide chain that are brought into spatial proximity due to the unique three-dimensional conformation of the enzyme protein.

The active sites of various enzymes incorporate radicals of specific amino acid residues, primarily the OH groups of Serine, Threonine, and Tyrosine, the imidazole ring of Histidine, the SH group of Cysteine, the COO^- groups of dicarboxylic Amino Acids, and the NH₃⁺ groups of Arginine and Lysine (Fig. 7.2). The Cofactors of a given enzyme—prosthetic groups and Metal Ions—also participate in The formation of active sites (Fig. 7.3).

Fig. 7.2. Three-dimensional Structure OF THE enzyme Chymotrypsin. The Active Site of the enzyme is formed by The amino acid residues Ser-195, His-57, and Asp-102.

Fig. 7.3. Location OF THE Zn atom in the active site of the enzyme Carbonic anhydrase.

The active site is typically localized within a cleft or spatial niche formed in the macromolecule of the enzyme protein. The structure of the active site is complementary to the spatial architecture of the substrate, which formed The basis of Emil Fischer's concept of enzyme and substrate matching as a "lock and key." Later theories of enzymatic catalysis (such as D. Koshland's) account for possible mutual Changes in the spatial Conformations of the enzyme and substrate during their interaction (the "induced fit theory of enzyme and substrate").

The structure of the active site comprises:

- the substrate-binding site, or contact ("anchor") site; it contains radicals of polar (which bind substrate molecules via Hydrogen Bonds or dipole interactions) or nonpolar amino acid residues (forming hydrophobic zones in the active site that interact with corresponding radicals in the substrate);

- the catalytically active site, which includes chemical groups that directly participate in the transformation of the substrate (-OH, -SH, ≥N, -NH₃⁺, -СОО^- groups).

MECHANISMS OF SUBSTRATE transformation during enzymatic catalysis

The Mechanism of Enzymatic substrate transformation involves the following molecular effects:

1. Proximity and Orientation effects. Adsorption and physicochemical interaction of substrates with the active sites of enzymes are accompanied by a local increase in the concentration of reacting molecules, their bringing together, and their most effective orientation relative to each other and to the catalytically active groups of the active site.

2. Acid-Base Catalysis effects. Certain Functional groups of amino acid radicals that form the structure of active sites possess The properties of Brønsted acids or bases, i.e., proton Donors or acceptors (amino, carboxyl, sulfhydryl, and imidazole groups).

An example of an acid-base catalyst is the imidazole group of histidine, which, through interaction with the hydroxyl group of serine, forms a Brønsted acid-base pair. The functional groups ≥N and -OH that play a catalytic role are generally located in different regions of the peptide chain and are brought into close proximity due to the unique Tertiary Structure of the enzyme protein.

3. Nucleophilic and electrophilic catalysis effects. Functional groups of amino acid residues within active sites can act in the catalytic act of substrate transformation as electron donors (nucleophiles) or electron acceptors (electrophiles), i.e., as Lewis bases or acids, respectively.

Nucleophilic groups within the active sites of enzymes:

Electrophilic groups within the active sites of enzymes:

- NH₃⁺ — groups of arginine and lysine;

- cofactor metal ions (Mg²⁺, Fe³⁺, Mn²⁺, Cu²⁺).

Mechanisms of chymotrypsin catalytic action

The mechanisms of enzymatic action are best studied for Hydrolases, which are catalysts with acid-base type active sites. Examples of such enzymes include biocatalysts whose active sites contain histidine-serine catalytic complexes; these include widely known enzymes such as chymotrypsin (EC 3.4.4.5), Trypsin (EC 3.4.4.4), Thrombin (EC 3.4.4.13), Elastase (EC 3.4.21.11), and acetylcholinesterase (EC 3.1.1.7).

Chymotrypsin (α-chymotrypsin) is a proteolytic enzyme (protease) that cleaves peptide bonds involving Water molecules in certain Proteins AND Peptides. As noted earlier (Fig. 7.2), the catalytic center of chymotrypsin is formed by the hydroxyl group of serine and the imidazole group of histidine, located at positions 195 (Ser-195) and 57 (His-57) of the peptide chain of the enzyme molecule, respectively.

First stage of the catalytic act.

Imidazole acts as a Brønsted base, withdrawing a proton from the OH group of serine, which leads to an excess electron density on the serine oxygen atom and facilitates the nucleophilic attack on the -CO- group of the substrate by the serine hydroxyl. As a result of this process, the enzyme is acylated through The transfer of the acyl radical from the substrate to the serine residue (Ser-195) of the enzyme (Fig. 7.4 a, b).

Second Stage of the catalytic act.

The histidine atom (His-57) performs a nucleophilic attack on the oxygen of the serine acyl derivative, which facilitates the Cleavage and transfer of the acyl group to an external acceptor—a water molecule (Fig. 7.4 c, d).

Fig. 7.4. Involvement of the functional groups of the chymotrypsin active site (His-57 and Ser-195) in the catalytic Cleavage of the peptide bond in the substrate molecule.

Mechanisms of acetylcholinesterase catalytic action

Acetylcholinesterase (EC 3.1.1.1) is an enzyme that hydrolytically cleaves the neurotransmitter acetylcholine molecule:

Acetylcholinesterase also belongs to histidine-serine hydrolases and catalyzes the hydrolytic cleavage of the substrate via the transient formation of an acetylated serine residue intermediate. Furthermore, the active site of acetylcholinesterase includes a tyrosine hydroxyl group which acts as a base catalyst, facilitating the Hydrolysis of the acetylated serine and the regeneration of the original active site structure of the enzyme.