STRUCTURE AND PROPERTIES OF BIOMOLECULES - A. E. Zemlyakov - 2017

03. ENZYMES

Virtually all metabolic processes in living organisms proceed with the participation of protein compounds. Among the most vital Functions of Proteins are enzymatic activity, as well as roles in the hormonal and immune systems. Proteins are also classified into distinct groups such as transport, structural, motor, regulatory, and many others. This chapter explores the enzymatic Properties of Proteins, while various other Protein Functions will be examined in subsequent chapters.

❖ Enzymes (from Lat. fermentum - leaven) are proteins or Structure/178.html">Protein Complexes that act as specific and highly efficient catalysts for Chemical Reactions occurring within living Cells. The term enzyme (from Greek évzyme - leaven) is used as a synonym.

The term "ferment" (enzyme) was introduced into scientific practice in the early 17th century by the Dutch scientist Jan Baptista van Helmont to designate substances that facilitate Digestion.

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In the second half of the 19th century, the outstanding French microbiologist and chemist Louis Pasteur suggested that Fermentation is catalyzed by a certain vital force residing within Yeast cells. Later, in 1897, the German chemist and biochemist E. Buchner experimentally proved that fermentation can also be activated by Cell-free yeast juice. In 1907, he was awarded the Nobel Prize in Chemistry for his discovery of cell-free fermentation.

The definitive proof of the protein nature of enzymes is associated with the American biochemist and 1946 Nobel laureate in Chemistry, J. Sumner, who obtained the enzyme urease in crystalline form in 1926.

Enzymes span a wide range of molecular weights, from 10 kDa to over 2 MDa. Along with polypeptide chains, their composition may include non-protein components (Cofactors) such as Metal Ions, Vitamin-like compounds, and Porphyrins.

Table 2. Metal cations as enzyme cofactors

The functioning of certain enzymes requires the presence of specific organic molecules known as Coenzymes. Typically, they facilitate The transfer of particular chemical groups or ions (see Table 3).

Table 3. Groups transferred with the participation of coenzymes

A coenzyme that is tightly bound to a protein is referred to as a prosthetic group. If the "coenzyme-protein" complex is readily dissociable, the complete complex is called a holoenzyme, while the protein component alone without its coenzyme is termed an apoenzyme.

When an enzyme (Е) interacts with a substrate (S), an intermediate enzyme-substrate complex (ES) is formed, which subsequently dissociates to yield the reaction product (Р) and regenerates the free enzyme.

The interaction with the substrate takes place in a specialized region of the enzyme known as the Active Site. The substrate Specificity of an enzyme is ensured by several structural fragments (binding sites) that facilitate substrate recognition and anchoring at multiple points (for details, see below).

✵ Enzyme Selectivity. Over 4,000 Reactions Catalyzed by proteins are currently known. Enzymes are highly specific both with respect to the type of reaction they catalyze (chemoselectivity) and to their substrates. The enzyme-substrate binding constant can reach as high as 10^-10 mol/L. Furthermore, enzymes exhibit regioselectivity, catalyzing reactions at specific functional groups. For instance, the action of two different Decarboxylases on aspartic acid selectively cleaves either the α- or β-carboxyl group.

Many enzymes are stereospecific, demonstrating both enantioselectivity toward asymmetric centers of the substrate and diastereoselectivity regarding Cis- and trans-configurations. For example, the enzyme fumarase catalyzes the reversible conversion of fumarate to L-malate, yet it shows no activity toward either maleate or D-malate.

✵ The catalytic efficiency of enzymes in biochemical reactions is exceptionally high. For example, at 37 °C, a single molecule of the enzyme rennin coagulates ~10⁵ molecules of milk caseinogen within 1 min. Certain enzymes are so active that the reaction occurs virtually upon every contact with the substrate, meaning the reaction rate is limited solely by The rate of substrate diffusion. Such enzymes are termed catalytically perfect enzymes; notable Examples include acetylcholinesterase, fumarase, β-lactamase, and superoxide dismutase.

✵ Units of enzymatic activity. Enzymatic activity is commonly expressed in International Units (IU), defined as The amount of enzyme that catalyzes The conversion of 1 µmol of substrate to product per 1 min, or in katals (kat), defined as the amount of catalyst that converts 1 mol of substrate to product per 1 sec. 1 kat = 6·10⁷ IU.

❖ Enzyme Nomenclature. Typically, an enzyme's name consists of the substrate name combined with the suffix -ase. For example, arginase catalyzes the Hydrolysis of Arginine, Ribonuclease cleaves RNA, and so on.

Alternatively, enzyme names are formed by adding the suffix -ase to the name of the catalyzed reaction. For instance, dehydrogenase catalyzes the removal of hydrogen, hydrolase catalyzes hydrolysis, and so forth. Several enzymes have retained their traditional trivial names, such as Chymotrypsin, Papain, and Lysozyme.

❖ Enzyme Classification.

✵ Based on structural Organization:

♦ Enzymes consisting of a single polypeptide chain (e.g., lysozyme).

♦ Enzymes consisting of multiple polypeptide chains linked by Disulfide Bonds (e.g., chymotrypsin).

♦ Enzymes composed of multiple subunits joined by non-covalent bonds (e.g., Lactate dehydrogenase).

♦ Polyfunctional enzyme assemblies — a single polypeptide chain forms the active sites of several functionally related enzymes. For example, three enzymes involved in pyrimidine Biosynthesis—carbamoyl phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase—are integrated into a single polypeptide chain with a Molecular Weight of 2.15 MDa.

♦ Multienzyme complexes — a series of sequentially acting enzymes linked through non-covalent interactions (or associated in a complex) to catalyze consecutive reactions; for instance, FATTY ACID BIOSYNTHESIS is carried out by a complex of 7 enzymes.

✵ Based on the type of catalyzed reaction:

♦ oxidoreductases — catalyze oxidation-reduction processes,

♦ transferases — catalyze the transfer of functional groups,

♦ Hydrolases — catalyze hydrolysis reactions,

♦ lyases — catalyze addition across double bonds,

♦ isomerases — catalyze isomerization reactions,

♦ ligases — catalyze synthetic reactions driven by ATP energy.

❖ Main classes of enzymes. In 1972, a new international system of enzyme nomenclature was adopted, classifying enzymes According to the Nature of the reaction they catalyze. Each enzyme is designated by a sequence of numbers. The first digit represents the main class, the second refers to the subclass detailing the reaction type, and subsequent digits provide further specifications. For example, 2.1.1 designates

2 — the class of transferases,

2.1 — transfer of a single-carbon group,

2.1.1 — transfer of a methyl group.

✵ 1. Oxidoreductases — enzymes that catalyze oxidation-reduction reactions. These enzymes transfer hydrogen atoms (or electrons) and facilitate the hydrogenation and dehydrogenation of Biomolecules. They typically incorporate specific coenzymes.

For example, alcohol dehydrogenase reversibly oxidizes alcohols to carbonyl compounds.

✵ 2. Transferases — enzymes that catalyze the transfer of chemical groups, such as methyl, carboxyl, formyl, or phosphate groups, between molecules.

The transfer is carried out by specific carriers that act as coenzymes. For example, protein Kinases catalyze the phosphorylation of proteins utilizing the phosphate group of ATP.

✵ 3. Hydrolases – enzymes that catalyze hydrolytic Cleavage reactions. They are named according to the type of bond they cleave (e.g., glycosidases, esterases, etc.).

A classic example is amylase, an enzyme that hydrolytically cleaves the polysaccharide amylose.

✵ 4. Lyases – enzymes that catalyze The addition of groups to double bonds, or conversely, the non-hydrolytic removal of groups or molecules (including СО₂, Н₂О, NНз, and other more complex ones) with The formation of a double bond.

The action of fumarase, an enzyme belonging to this class, was discussed above.

✵ 5. Isomerases – enzymes that catalyze isomerization reactions, including racemization, shifting of double bonds, rearrangement of groups around an asymmetric carbon atom, shifting of a phosphate group to another carbon atom, and so on.

For instance, Triosephosphate isomerase catalyzes the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.

✵ 6. Ligases (synthetases) – enzymes that catalyze the Condensation of two molecules, coupled with the hydrolysis of a high-energy bond in ATP or another nucleoside triphosphate. For example, a key step in Replication—joining DNA fragments into a continuous strand—is carried out by DNA ligase.

❖ Factors affecting the Rate of Enzymatic reactions.

✵ Medium pH. Changes in the pH of the medium affect the conformation of the enzyme and, consequently, its catalytic activity. Each enzyme has an optimum pH; for instance, the optimum pH for Pepsin is 1.5, while for arginase it is 9.5–9.9.

✵ Temperature. According to the van 't Hoff rule, the rate of an enzymatic reaction roughly doubles with a 10 °C temperature increase, typically up to 40–50 °C. Higher temperatures can cause Protein Denaturation. At the same time, Bacteria have been discovered living in high-temperature hydrothermal vents in the deep ocean ("black smokers"). Consequently, their enzyme systems can withstand significantly higher temperatures.

✵ Activators – substances that increase enzyme activity.

✵ Inhibitors – substances that slow down or prevent enzyme action. They are divided into irreversible inhibitors, which permanently modify crucial sites on the enzyme by forming covalent bonds, and reversible inhibitors, which interact with the enzyme without forming covalent bonds. Reversible inhibitors can be removed by washing, dialysis, or Gel filtration, thereby restoring enzyme activity.

Specifically, diisopropylfluorophosphate is often used as an irreversible inhibitor for Serine-containing enzymes, whereas iodoacetic acid is used for Cysteine-containing enzymes.

The interaction between an enzyme and a reversible inhibitor can, in turn, be classified as competitive or non-competitive inhibition. In the former case, the inhibitor (I) structurally resembles the substrate and binds to the same active site. Typically, no catalytic reaction takes place with the inhibitor. Such inhibitors bind less tightly to the enzyme, and enzyme activity can be restored by increasing the Substrate Concentration.

In non-competitive inhibition, the inhibitor (I*) differs structurally from the substrate and binds to the enzyme at a site distinct from the substrate-binding site. Typically, this type of inhibitor does not prevent the Formation of the enzyme-substrate complex, but rather disrupts the catalytic process of converting the substrate into the reaction product. In this case, increasing the substrate concentration does not restore enzyme activity.

❖ Active site. The substrate binds to the enzyme to form a complex within a specific region known as the active site. The active site is frequently formed by amino acid residues that are spatially distant in the Primary Structure. It is conventional to distinguish between the binding site and the catalytic site.

The catalytic site is typically formed by amino acid residues that are separated from each other in the primary structure. When the peptide chain folds into its tertiary structure through valence and non-valence interactions, these Amino Acids are brought into spatial proximity. The figure illustrates how the convergence of non-polar side chains via hydrophobic interactions creates the Active Site of the enzyme (the constituent amino acids are indicated by arrows).

Binding determines Enzyme Specificity. Various substrate fixation principles may be involved. For example, in ribonuclease, the heterocyclic base forms Hydrogen Bonds with Serine and Threonine residues; in chymotrypsin, hydrophobic amino acids are anchored in a non-polar "pocket" through hydrophobic interactions; and in Trypsin, basic amino acids (Lys or Arg) are secured via ionic interactions.

In 1890, Emil Fischer proposed the "lock-and-key" hypothesis of enzyme action, which suggests that There is a precise geometric correspondence between the shapes of the substrate and the enzyme.

In 1958, Daniel Koshland put forward the "induced-fit" (hand-glove) hypothesis of enzyme action. During substrate recognition and binding, a conformational adjustment of the enzyme to the substrate takes place—involving the precise orientation of catalytic and binding groups, and the generation of specific strains within the substrate structure that makes it more susceptible to the catalyst. The catalytic region comprises amino acids that directly participate in catalysis, which typically possess ionogenic groups.

Substrate binding is generally driven by non-covalent interactions, such as hydrogen bonds, ion-ion, dipole-dipole, and hydrophobic interactions. In some instances, covalent bonds are formed, such as the formation of acyl-enzymes (see chymotrypsin below). The process of substrate fixation induces Conformational Changes in the Enzyme Structure, causing it to "mold" to the substrate structure. Concurrently, multi-center binding introduces structural strain into the substrate, thereby facilitating the chemical reaction at the active center.

❖ Allosteric enzymes. The activation of these enzymes requires prior interaction with a specific Ligand known as an effector (activator, A). This interaction occurs at a specialized site on the enzyme called the regulatory center. The formation of the enzyme-effector complex induces a structural change in the enzyme, enabling it to interact with the substrate.

Allosteric inhibition can likewise be reversible or irreversible. In this case, the allosteric inhibitor (AI) binds to the regulatory center and either competes with the effector for binding or, through chemical interaction, renders the effector unrecognizable. In both scenarios, structural alterations in the enzyme preclude interaction with the substrate.

❖ Enzyme activation. Many enzymes are synthesized in the body as inactive precursors known as proenzymes. One mechanism for their conversion into active forms involves the covalent modification of functional groups on amino acid residues, which alters the biomolecule's conformation and leads to the formation of the active site.

Most frequently, this involves the phosphorylation or adenylylation of hydroxyl groups mediated by adenosine triphosphate (ATP), as well as the methylation of carboxyl groups by S-adenosylmethionine (Met-A).

Another activation mechanism involves the proteolytic cleavage of a portion of the peptide chain by another enzyme. For instance, a hexapeptide fragment is cleaved from the polypeptide chain of inactive trypsinogen by an enzyme, yielding the active enzyme trypsin. Trypsin, in turn, participates in the activation of chymotrypsin (see below).

❖ Lysozyme (muramidase) is an enzyme that hydrolyzes the polysaccharide of bacterial cell walls (murein). It was discovered in 1922 by the English biochemist Alexander Fleming and belongs to the class of glycosidases. It is found in various PLANT AND ANIMAL Tissues, notably in tears and egg white. In cellular tissues, lysozyme localizes in Lysosomes.

Over 50 muramidases from various sources have been isolated and characterized. Lysozymes are species-specific; for example, hen egg-white lysozyme contains 4 S-S bonds and 6 Tryptophan residues, whereas human lysozyme contains 3 S-S bonds and 5 tryptophan residues. Lysozyme is rich in basic amino acids, which facilitates its interaction with the negatively charged Introduction/37.html">Bacterial Cell wall.

The molecular mass of hen egg-white lysozyme is 14.6 kDa, and it consists of 129 amino acids. The enzyme is an ellipsoid with axes of 3 and 4.5 nm, divided into two domains: one is flexible and rich in polar amino acids, while the other is rigid, with an elevated content of hydrophobic amino acids. Approximately 25% of the polypeptide chain's amino acid residues are incorporated into α-helices.

A cleft situated between the two halves of the lysozyme molecule accommodates the hexasaccharide fragment of peptidoglycan, where the cleavage process takes place. At least 12 amino acids are involved in binding. The active site comprises two acidic amino acids, Glu-35 and Asp-52. The former resides in a non-polar environment, rendering its carboxyl group unionized, whereas the latter is situated in a polar environment, and its carboxyl group is accordingly ionized.

One of the proposed mechanisms of lysozyme action is based on the hypothesis that, during polysaccharide binding at the catalytic site, the muramic acid residue (fragment D) cannot adopt the usual chair conformation. The carbohydrate ring shifts into a less stable half-twist conformation, introducing steric strain around the glycosidic center.

The glycosidic oxygen atom is protonated by Glu-35, leading to the Cleavage of the glycosidic bond. As a result, the hexasaccharide splits into two fragments containing four and two carbohydrate residues, respectively. This breakdown of the muramine polysaccharide chain disrupts the bacterial cell wall, which underlies the antibacterial action of lysozyme.

Lysozyme is used in medicine as an antiseptic and anti-inflammatory agent. Specifically, it enhances the efficacy of β-lactam Antibiotics, such as penicillin.

❖ Chymotrypsin is a proteolytic enzyme (endopeptidase). Its primary function is the Hydrolysis of Proteins and Peptides.

Cleavage occurs predominantly at aromatic amino acids (Tyr, Trp, Phe) as well as Met. This enzyme is also capable of catalyzing the transfer of aromatic acyl groups to Other Amino Acids, making it useful in enzymatically catalyzed syntheses.

The molecular weight of chymotrypsin is ~25 kDa. Its molecule consists of three chains (A, B, and C) containing 241 amino acids linked by 5 S-S bonds. The C-terminus of chain C has an α-helical structure. Its overall shape is ellipsoid.

Chymotrypsin is secreted as a proenzyme, chymotrypsinogen. Its conversion into the active enzyme takes place in the duodenum through the action of trypsin, which excises two dipeptides, (Ser-14 - Arg-15) and (Thr-147 - Asn-148) (highlighted in purple in the figure). This triggers a rearrangement of the three chains to form the catalytic center.

The substrate binds to the enzyme via hydrophobic interactions. The active site of the enzyme comprises Three amino acids: Ser-195, His-57, and Asp-102.

During catalysis, a nucleophilic attack is carried out by the oxygen atom of the serine side chain on the carbonyl carbon of the peptide chain, forming a covalent bond between the substrate peptide residue and The amino acid residue. This enzyme intermediate is called an acyl-enzyme, which then undergoes hydrolysis. In these processes, aspartic acid acts as a proton donor/acceptor, while the heterocyclic moiety of Histidine functions as a proton shuttle.

Respiratory Enzymes

A number of protein molecules contain non-protein components—specifically pigments and metal ions—and are therefore classified as Chromoproteins and Metalloproteins. Other types of protein complexes also occur in nature, such as Glycoproteins (proteins containing one or more oligosaccharide chains), Lipoproteins (spherical protein-lipid micelles), and Nucleoproteins (Complexes of Proteins with DNA or RNA).

This section focuses on the Structure and function of chromoproteins specialized in the reversible binding and transport of oxygen to Organs and tissues. Such proteins are often referred to as respiratory enzymes. The structure and function of another chromoprotein, rhodopsin, will be discussed in the next chapter.

❖ Myoglobin is a chromoprotein found in tissues, specifically in skeletal and cardiac Muscle, that facilitates Oxygen transport from Blood Hemoglobin to the Cytochromes of muscle cells. Muscle Tissues of marine mammals are particularly rich in myoglobin. For instance, myoglobin accounts for 3.5% and 7.7% of the muscle mass in dolphins and seals, respectively.

The polypeptide chain of sperm whale myoglobin contains 153 amino acid residues, with a molecular weight of ~17 kDa. Its structure features 8 helical regions designated by the letters A through H. Enclosed within the protein globule (measuring 4.5 x 2.5 nm) is a porphyrin structure known as the heme group, which contains a coordinately bound Fe²⁺ ion.

The heme is anchored within the molecule through hydrophobic interactions with the polypeptide chain, as well as a coordination

bond between the iron atom of the heme and the nitrogen atom of a histidine residue in the F helix. On the other side of the heme, near the iron atom, lies a histidine nitrogen atom from the E helix, though at a greater distance.

An oxygen molecule can reversibly enter this hydrophobic "pocket," coordinating simultaneously with both the iron atom and a nitrogen atom to form oxymyoglobin. Because this region has low polarity, The oxidation of Fe²⁺ to Fe³⁺ is prevented.

The release of the oxygen molecule from oxymyoglobin occurs During Muscle contraction, when capillary compression causes a sharp drop in the partial pressure of oxygen. The protein acts as a Water-soluble carrier for heme, protects Fe2+ from oxidation, and regulates oxygen affinity.

❖ Hemoglobin. Red Blood Cells contain hemoglobin protein molecules (each cell holds ~340 million molecules) responsible for oxygen transport throughout the body. This chromoprotein consists of two α- and two β-subunits, containing 141 and 146 amino acids, respectively. Each protein component, structurally similar to myoglobin, contains a heme molecule (shown in red) capable of reversibly binding one oxygen molecule.

In 1962, the Nobel Prize in Chemistry was awarded to British researchers J. Kendrew and M. Perutz for their research into the Spatial Structure of Myoglobin and hemoglobin.

Each subunit performs its biological function in interaction with the others. The first oxygen molecule binds to the α-subunit, which undergoes a conformational change as a result.

These conformational changes are transmitted to the β-subunit, facilitating its binding of an oxygen molecule. The "oxygenated" αβ-subunit block alters the conformation of the second αβ-block, leading to its oxygenation.

This cascade of conformational changes results in the overall conformation of the oxygen-bound hemoglobin molecule (oxyhemoglobin) differing from that of the oxygen-free molecule (deoxyhemoglobin).

✵ Sickle-Cell Anemia.

Some individuals, particularly those living in South and Southeast Asia, suffer from a blood disorder known as sickle-cell anemia, which is associated with altered structures of hemoglobin and, consequently, erythrocytes. In patients with this condition, the sixth amino acid in the β-subunit of hemoglobin is valine instead of glutamic acid. Even such a minor alteration in Amino Acid Composition leads to significant changes in the spatial structure of hemoglobin. Unlike normal hemoglobin A, this hemoglobin S has a reduced capacity for oxygen transport. Patients exhibit symptoms of Hypoxia, or oxygen deprivation. Red blood cells carrying hemoglobin S have an altered, sickle-like shape—hence the name of the disease. They exhibit reduced stability and are more prone to destruction.

❖ Hemocyanin. In Mollusks and Arthropods, the oxygen transport function is performed by the metalloprotein hemocyanin. The functional domain in mollusks consists of two regions: α and β. A hemocyanin subunit can contain 7–8 molecular functional domains, and its mass reaches 550 kDa.

The α-helical region contains two copper atoms, each coordinated with three histidine molecules. An oxygen molecule is accommodated in the cavity between the copper atoms, acting as the fourth coordination center. In this process, the distance between the metal atoms decreases from 0.46 to 0.36 nm.