Pharmacognosy with Basics of Plant Biochemistry - Kovalyov V. M. 2004

Special Part
Peptides and Proteins
Enzymes

Ferments, or Enzymes, are biological catalysts of a protein nature present in all living Cells. They take part in biochemical transformations, thereby directing and regulating the METABOLISM within the Organism.

The name "ferment" originates from the Latin fermentum, meaning leaven, which is associated with The Nature of Fermentation processes. Initially, the term "ferment" was used to define living microorganisms involved in fermentation, while "ferment-substances" such as Pepsin, emulsin, and others were designated by the term "enzyme" (from the Greek enzym — in leaven). However, it was later demonstrated that an enzyme preparation could be isolated from Yeast cells and that Enzymatic Catalysis can occur outside a living organism. Thus, the need to use two separate terms, "enzyme" and "ferment," to describe the same concept disappeared, though both names remain widespread and are now considered synonyms.

Enzymes are used in various sectors of the national economy, but the proportion applied in medicine is relatively small. They are characterized by a high degree of purification and complex, costly production technologies. Out of the 3,000 enzymes currently known, about 40 are used in the medical and microbiological industries of the CIS countries for drug manufacturing. Of these, 62% are animal-derived preparations, 33% are derived from microbial cultures, and only 5% come from plant raw Materials. It should be noted that the current level of development and Structure/175.html">Implementation of medicinal enzyme preparations in CIS countries fails to fully satisfy the existing demand. Overall, the arsenal of enzymes used in clinical practice accounts for only 10% of all scientifically known enzymes with a proven therapeutic effect.

Structure and Classification

Structural features. The protein nature of enzymes has been confirmed by X-Ray Diffraction Analysis. In terms of Amino Acid Composition, enzymes do not differ from other Proteins, exhibiting the same four levels of molecular structural Organization. Most often, enzymes are composed of two or more Peptides linked non-covalently. However, the presence of all four orders of macromolecular structure is not always mandatory. Simpler enzymes, such as Lysozyme, Trypsin, and Ribonuclease, lack a quaternary structure.

Both simple and complex enzymes exist in nature. The former consist entirely of Polypeptides and break down exclusively into Amino Acids upon Hydrolysis (these include pepsin, trypsin, Papain, urease, and lysozyme, among others). Most enzymes belong to the Class of Conjugated Proteins, which contain a non-protein component—a cofactor—whose presence is essential for enzymatic activity. The polypeptide part of a conjugated enzyme is conventionally called the apoenzyme. Conjugated enzymes with a small dissociation constant that do not dissociate into apoenzyme and cofactor during purification and isolation are termed holoenzymes, while the cofactor is known as the prosthetic group. A coenzyme is typically understood to be a cofactor that easily dissociates from the apoenzyme. The same cofactor can function as both a prosthetic group and a coenzyme. Vitamins are classic representatives of Coenzymes. Certain divalent metals (Ca²⁺, Mg²⁺, Mn²⁺) also act as Cofactors. The cofactor function of BIOLOGICALLY ACTIVE SUBSTANCES such as HS-Glutathione, ATP, linoleic acid, nucleoside derivatives, and porphyrin-containing compounds has likewise been established.

When studying the Mechanism of Enzymatic reactions, it was observed that substrate molecules are typically small compared to enzyme molecules. This led to the hypothesis that upon The formation of enzyme-substrate complexes, only a limited number of polypeptide amino acids come into contact with the substrate molecule—the so-called active center. The active center is understood as a unique combination of amino acid residues that ensures direct interaction with the substrate and participates in the catalytic reaction. It has been established that prosthetic groups are incorporated into the active center. Conventionally, the active center comprises a catalytic center, which enters into chemical interaction with the substrate, and a binding center (or contact/"anchor" site), which ensures specific affinity for the substrate and the formation of its complex with the enzyme. The active center determines the catalytic activity and Specificity of the enzyme, while the conformation of the entire molecule is crucial for its action. Structural disruption (such as Denaturation) causes partial or complete destruction of the active center and, consequently, the loss of the enzyme's catalytic properties.

Classification. According to the classification adopted by the Enzyme Commission of the International Union of Biochemistry, all enzymes are divided into six classes based on the type of reaction they catalyze.

Oxidoreductases are enzymes that catalyze oxidation-reduction reactions and electron transfer.

Transferases are enzymes that catalyze The transfer of various functional groups from one substrate (donor) to another (acceptor).

Hydrolases are enzymes that catalyze the Cleavage of intramolecular bonds in substrates with The addition of Water according to the scheme:

R—R'+ НОН → RH + R'OH

Lyases are enzymes that catalyze the cleavage of bonds, including double bonds, without the addition of water.

Isomerases are enzymes that catalyze isomerization reactions.

Ligases (synthetases) are enzymes that catalyze biosynthetic processes involving the joining of molecules utilizing ATP energy.

Each of the six enzyme classes is subdivided into subclasses, which in turn are divided into sub-subclasses. According to modern classification, an individual enzyme has a name and a code number. In the code, the first digit designates the class, the second the subclass, the third the sub-subclass, and the fourth the specific enzyme.

Distribution and Localization of Major Types

The class of hydrolases (hydrolytic enzymes) includes the majority of enzymes with significant industrial or medical Applications.

The subclass of esterases (code 3.1.) comprises enzymes that catalyze the cleavage and synthesis of esters in accordance with the equation:

R—СОО—R₁ + Н₂O ⇆ R—СООН + R₁—ОН

Among esterases, lipases deserve special mention as they catalyze the cleavage and synthesis of fats. In the bodies of humans and animals, the most active lipase is found in pancreatic juice. The ability to cleave fats is also observed in many plants. Lipase is present in the seeds of cereals and oilseed crops such as soy, sunflower, cotton, and flax. At the same time, stinging nettle seeds are poor in lipase, which is located primarily in the Vegetative Organs of the plant. The potential action of lipase must be taken into account when storing plant raw materials containing significant amounts of oil. Elevated humidity and Temperature activate lipase, leading to fat cleavage with the formation of glycerol and free Fatty acids.

Another member of the esterase group is tannase, an enzyme that catalyzes the hydrolysis of tannin. It exhibits exceptional reaction specificity, cleaving only those esters whose acid component contains at least two phenolic hydroxyl groups.

Carbohydrases (code 3.2) are enzymes that catalyze the hydrolysis and synthesis of homo- and heteroglycosides.

Carbohydrases include a- and ß-amylases. Amylases break down starch into dextrans and maltose, which is the end product of complete starch hydrolysis by these enzymes. The most active amylases are found in Human and Animal saliva and pancreatic juice. Interestingly, plant seeds vary in their a- and ß-amylase content. For instance, ungerminated grains of wheat, rye, and barley contain only ß-amylase; a-amylase is formed in them only after germination. Soybean seeds contain ß-amylase in both germinated and ungerminated states.

ß-Fructofuranosidase (invertase or sucrase) catalyzes The breakdown of sucrose into glucose and fructose. This enzyme hydrolyzes the bond located at the ß-glucosidic carbon atom of the fructose residue, whereas a-glucosidase hydrolyzes the bond in sucrose at the a-glucosidic carbon atom of the glucose residue:

ß-Fructofuranosidase is found in higher plants, Yeasts, microorganisms, animal and human digestive juices, and flower pollen.

Proteases (EC 3.4), also known as peptide hydrolases, are enzymes that catalyze the hydrolysis of peptide bonds in proteins and polypeptides according to the following equation:

Proteases are divided into two groups: proteinases (Endopeptidases) and peptidases (exopeptidases). Proteinases hydrolyze proteins into polypeptides, whereas polypeptides are further cleaved by peptidases into amino acids.

Among proteinases, digestive enzymes such as pepsin, trypsin, and Chymotrypsin deserve special mention.

Pepsin is secreted by the gastric mucosa. It can be obtained in the form of protein crystals with a Molecular Weight of 34,644. The pepsin molecule consists of a single polypeptide chain comprising 327 amino acid residues. Its optimal activity is within the pH range of 1.2–1.5. In addition to proteins, pepsin is capable of hydrolyzing polypeptides and dipeptides. It has been established that it preferentially cleaves only those peptide bonds whose formation involves the amino groups of Tyrosine and phenylalanine. In the Cells of the gastric mucosa, pepsin exists as an inactive precursor, pepsinogen. Under the action of free pepsin and Hydrochloric acid, pepsinogen is converted into active pepsin.

Trypsin is present in pancreatic juice. Its molecular weight is 24,000, and the molecule consists of 229 amino acid residues. The optimal activity zone corresponds to pH 8–9. The degree of Protein Hydrolysis by crystalline trypsin is roughly comparable to that of crystalline pepsin; however, crude trypsin exhibits significantly higher catalytic activity. Crystalline trypsin catalyzes the hydrolysis exclusively of peptide bonds involving the carboxyl group of Lysine or Arginine. Trypsin is formed from inactive trypsinogen through the action of enterokinase, after which trypsin itself facilitates The conversion of trypsinogen into trypsin.

Pancreatic juice also contains an inactive enzyme, chymotrypsinogen. Upon exposure to traces of trypsin, it is converted into active chymotrypsin, a protein composed of 242 amino acids. The optimum activity is observed at pH 7.5–8.5.

Proteinases are also found in certain plants, with papain being a typical representative. It is extracted from the latex of the papaya tree. The optimal activity zone of the enzyme lies within weakly acidic, neutral, and weakly alkaline media (depending on the Nature of the substrate protein). The action of papain and other plant-derived Proteolytic Enzymes is activated by hydrocyanic acid and sulfhydryl compounds containing an SH-group, primarily Cysteine and reduced glutathione.

Papain-type proteinases have been found in the fruits and stems of the pineapple (Ananas comosus) as bromelain, as well as in the latex of plants of the genus Ficus, yielding the enzyme ficin.

Amidases (EC 3.5). This group of hydrolytic enzymes includes urease, which breaks down urea into ammonia and carbon dioxide:

CO(NH₂)₂+ Н₂О = СО₂ + 2NH₃

Urease is found in plants, Molds, and certain Bacteria. High concentrations of urease are present in soybean seeds and lily of the valley (Convallaria majalis). Watermelon seeds serve as a promising source for obtaining this enzyme.

Physical and Specific Properties

Most enzymes are Globular proteins, with only a few being fibrous (for example, Myosin). In shape, globular enzymes resemble "ellipsoids of revolution." The molecular weight of enzymes typically ranges from 10⁴ to 10⁶, with enzymes having a molecular weight of 20,000–60,000 being the most common. Molecular dimensions are on the order of several tens of angstroms.

Like all proteins, enzymes exhibit amphotericity and electrophoretic mobility, are incapable of dialysis through semipermeable membranes, can bind significant amounts of water (undergo Hydration), and are easily precipitated from aqueous solutions by salts or organic Solvents (acetone, ethanol) while retaining their catalytic properties.

Enzymes differ from chemical catalysts by their exceptionally high catalytic efficiency (increasing reaction rates by a factor of 10¹⁰ to 10¹³) and specificity. It is customary to distinguish between absolute, absolute group, relative group, and optical specificity. Absolute specificity implies an affinity for a single substrate only. Absolute group specificity is manifested in action on substances that are structurally similar and share a specific bond type. Relative group specificity occurs when an enzyme exhibits specificity toward a bond between specific parts of a molecule while being completely indifferent to the Chemical Structure of the molecule itself. Optical specificity implies an enzyme's affinity for a single stereoisomeric form of a substrate.

Methods of Isolation and Activity Determination

Isolation. Enzyme preparations are classified according to their source into enzymes obtained from animal Tissues or human Blood Plasma; enzymes that are products of microbial vital activity; and plant enzymes.

Raw materials of animal origin may include: the Pancreas of slaughtered livestock (preparations of trypsin, chymotrypsin, ribonuclease, pantripin, pancreatin, etc.), the gastric mucosa (pepsin, acidin-pepsin, abomin, pepsidil), cattle Lungs (ingitryl), cattle Heart tissues (cytochrome c), cattle Testes (lidase, ronidase), natural gastric juice of dogs and horses (gastric juice), human blood plasma (fibrinolysin), and chicken egg white (lysozyme). Bee honey is also known to exhibit amylase activity.

The Use of animal raw materials involves the need to process large quantities of tissues from slaughtered livestock to obtain the required amount of enzymes, as well as the creation of special storage conditions for the raw materials. Obtaining enzyme preparations from microbial cultures—specifically molds, bacteria, yeasts, and actinomycetes—is a promising alternative. Products of microbial metabolism include preparations such as a-amylase, L-asparaginase, orase, solizym, streptokinase, and others. Microbial raw materials are more accessible, and microorganisms are capable of producing A wide variety of enzymes with diverse actions. Disadvantages of microbial raw materials include a large volume of preparatory work: selecting nutrient media for growing producer strains, maintaining optimal sterilization and cultivation regimes, and process monitoring.

Plant raw materials are also used to obtain enzymes: papaya latex, black cumin seeds, watermelon seeds, flower pollen, etc. In A number of cases, the advantages of this type of raw material are significant: its harvesting is technologically simpler, and the material can be dried, ground, compactly packaged, and stored for long periods without The Need for special conditions.

The Isolation and Purification of enzyme preparations depend on the individual CHARACTERISTICS OF THE raw material and are therefore not standardized.

The First stage in the isolation of intracellular enzymes of animal and plant origin involves the mechanical disruption of cells. The extraction of enzymes from microorganisms and subcellular Organelles presents a challenge due to the high rigidity of MICROBIAL Cell WALLS and the tight binding of enzymes to organelles. In such cases, specialized homogenization methods are employed: physical (high pressure, ultrasound, ionizing radiation, freeze-thawing), chemical (action of acids, alkalis, salts, organic solvents), enzymatic (action of lytic enzymes), and biological (inhibition of Cell wall Biosynthesis, phage action, Antibiotics).

Enzyme extraction is carried out using water, dilute acid or alkali solutions, Buffer solutions, or organic solvents (such as ethanol, acetone, dioxane, etc.). The extractant is selected individually. Step-by-step extraction using various extractants is also employed.

The resulting extracts contain impurities of ballast substances with various molecular weights. Low-molecular-weight compounds are removed by dialysis, while Lipids are extracted with organic solvents. Acid, alkaline, or thermal denaturation is used to render ballast proteins insoluble. Precipitation of inactive impurities using heavy metal salts is also applied.

Following preliminary purification, or sometimes without it, the extract is fractionated. Organic solvents (methanol, ethanol, isopropanol, acetone, dioxane, etc.) and salt solutions, such as ammonium sulfate, are used for enzyme precipitation. Various Chromatography methods (adsorption, ion-exchange, affinity) and Gel filtration on Sephadexes are employed to separate and concentrate enzyme proteins. Enzymes are also concentrated via ultracentrifugation.

The method and technique of crystallization are selected individually for each enzyme. However, a crystalline state is not a criterion of homogeneity: enzyme crystals may sometimes contain impurities of other substances.

The Structure of enzymes is determined using chemical modification, X-ray crystallography, and spectroscopy methods. There is also the Site-Directed Mutagenesis method, which is based on substituting amino acids in the protein part of the molecule using Introduction/32.html">Genetic Engineering techniques.

Determination of activity. Enzymes are standardized according to their activity. Determining enzyme activity in biological objects presents certain challenges. This is most often due to the fact that enzymes are present in tissues at very low concentrations. Enzyme activity is indicated by the reaction rate they catalyze under specific measurement conditions. At optimal temperature, medium pH, and full substrate saturation, the reaction rate is proportional to the Enzyme Concentration, which is determined by The rate of substrate depletion or the rate of reaction product formation.

To express enzyme concentration and quantitatively assess its activity, the Commission on Enzymes of the International Union of Biochemistry recommended the international standard unit E (or U). The unit of activity of any enzyme is taken as the amount that, under optimal conditions, catalyzes the conversion of 1 micromole of substrate per minute (μmol/min). In connection with the introduction of the International System of Units (SI), a new Definition of the enzyme unit has been proposed—the katal (kat, kat). A katal is the enzyme activity that causes a reaction rate of 1 mole per second (1 mol/sec). 1 E corresponds to 16.67 nanokatals (nkat). Both Units of Measurement are used interchangeably. Measurements of activity units are carried out at a temperature of 25 °C, optimum pH, and a Substrate Concentration exceeding the saturation concentration. Under these conditions, the rate depends solely on the enzyme concentration.

In Practical Work with enzymes, specific and molar activities are frequently used. Specific enzyme activity is the number of enzyme activity units per 1 mg of protein (or the number of katals per 1 mg of active protein). The number of substrate molecules converted by a single enzyme molecule per minute is referred to as the turnover number, or molar activity. Each enzyme has its characteristic molar activity. Carbonic anhydrase is the most active of all known enzymes, with a molecular activity of 36 million.

Biological effects and applications

Disruptions in human metabolic processes caused by the absence or altered activity of any enzyme can be genetically determined (enzymopathies) or result from inflammatory processes, trauma, tumors, surgical interventions, etc. The Treatment of diseases of various etiologies with enzymes is carried out through replacement therapy and the targeted use of enzymes in the pathological process.

Based on the experience of domestic and foreign enzymotherapy, six main areas for the use of medicinal enzyme preparations are conventionally defined.

Replacement therapy for gastrointestinal tract disorders. The use of enzymes in cases of their deficiency within the GI tract promotes nutrient absorption and normalizes secretory activity. For example, Triticase—a preparation based on a novel original plant substance developed at the SCACE (State Scientific Center for Medicinal Substances) from sprouted wheat seeds (Triticum vulgare, fam. Poaceae)—contains amylolytic enzymes (ß- and a-amylase), as well as ß-galactosidase, invertase, amino acids, and microelements. The granules are used as a replacement therapy agent with amylolytic and anti-inflammatory effects for the treatment of digestive disorders arising from enzyme functional insufficiency, particularly in Chronic Pancreatitis of various etiologies.

Treatment of acute and chronic inflammatory processes and wounds. Proteolytic enzymes hydrolyze inflammation-damaged tissue debris and purulent exudates, exhibiting anti-inflammatory, fibrinolytic, and wound-healing effects. Furthermore, enzymes can potentiate the action of antibiotics, increasing their concentration in the blood and affected tissues. The enzyme preparation lysozyme directly exerts a bacteriological effect by disrupting The Cell walls of microorganisms. Hyaluronidase preparations (lidase and ronidase) facilitate the resorption of scars and adhesions of various origins.

Enzyme therapy for cardiovascular diseases. Medications based on proteolytic enzymes improve capillary permeability in Blood Vessels, providing hypotensive and thrombolytic effects. Trypsin, chymotrypsin, terrilytin, fibrinolysin, streptokinase, streptodase, and celiase are used in the treatment of thrombosis. Cytochrome C is used to treat impaired tissue Respiration.

Comprehensive therapy for oncological diseases. Proteolytic enzymes have been used since the late 19th century to treat malignant tumors. Under the action of enzymes, the matrix connecting tumor cells to each other and to the endothelium is degraded, along with the proteolysis of Cancer cell membranes, leading to tumor shrinkage and necrosis. In enzyme therapy for malignant tumors, alongside proteases and Nucleases, the enzyme L-asparaginase is used; it catalyzes the hydrolysis of asparagine—an amino acid essential for cancer cell growth—into aspartic acid and ammonia. Asparagine depletion inhibits tumor cell growth and metastasis formation. L-Asparaginase is used to treat acute lymphoblastic leukemia.

Treatment of allergic conditions. The use of penicillin antibiotics carries the risk of allergic reactions and sometimes Shock states. In such cases, the use of the enzyme penicillinase is indicated. This penicillin antibiotic inactivator hydrolyzes their ß-lactam ring.

Use of enzymes as biochemical Reagents. The immobilized enzyme urease is used in the dialysis regeneration System of the artificial Kidney machine. By catalyzing the hydrolysis of urea, urease actively helps cleanse the blood of toxic substances.

Data on The activity of certain enzymes are presented in Table 2 of the Appendices.

Enzyme Activators and Inhibitors

Studies of enzymatic reactions occurring in living organisms have revealed that enzyme activity is determined by the presence of activators and inhibitors in the environment. The former accelerate the reaction, while the latter inhibit it. Various chemical substances can act as enzyme activators. For instance, hydrochloric acid activates pepsin, Bile acids activate pancreatic lipase, and papain is activated by compounds containing free SH groups (glutathione, cysteine). Metal Ions of di- or monovalent metals are particularly frequent activators. Nearly a quarter of all known enzymes require metals to exhibit full catalytic activity.

Enzyme Inhibitors are substances that cause partial or complete inhibition of enzyme activity. Any agents that cause Protein Denaturation also lead to Enzyme inactivation, but such inactivation is non-specific.

Specific inhibitors are of practical importance. They are used in enzymology to determine the nature of an enzyme's active center and its functional groups, to study The Mechanism of catalytic reactions, and generally to investigate metabolic processes within the organism. The action of many toxins is associated with Enzyme Inhibition. For example, poisoning by hydrocyanic acid occurs As a result of the inhibition of the respiratory enzyme cytochrome c oxidase. In recent years, enzyme inhibitors have found application in pathological processes characterized by excessive enzyme activity in the organism.

Practically all known inhibitors affect hydrolytic enzymes. Currently, medicine uses only animal-derived proteolytic inhibitors of polypeptide nature (pantrypsin, trielin, trasylol, kontrykal, gordox, etc.) or synthetic ones. A number of hydrolytic enzyme inhibitors, such as proteases, amylases, and lipases, have been found in plants of various families. The Biological Role of these inhibitors is not yet fully understood.

Plants of the Fabaceae, Poaceae, and Solanaceae families are rich in trypsin and chymotrypsin inhibitors. Inhibitor proteins are most commonly found in seeds and are localized in the aleurone grains, nuclei, Chloroplasts, and Mitochondria of cells. In some plants, they have been found in leaves, stems, flowers, tubers, etc. Scientists at the SSCL (State Scientific Center for Medicines) isolated a protease inhibitor from soybean seeds (Glycine max, Fabaceae). Its potential application as a medicinal product is currently being studied. As a result of their research, the SSCL has also developed a Technology for Obtaining a protease inhibitor—inamil—from wheat seeds. This preparation can be used for the clinical Diagnosis of gastrointestinal tract diseases and as a hypoglycemic agent for treating Carbohydrate Metabolism disorders.

Amylase inhibitors have been found in plant storage organs, as well as in leaves, fruits, and seedlings. They are represented by proteins or Phenolic Compounds, specifically Tannins. Species of the Fabaceae, Poaceae, and Theaceae families are rich in amylase inhibitors.

Natural lipase inhibitors isolated from plants belong to the class of proteins or lipids and exhibit high activity against pancreatic lipases. Inhibitors of lipolytic enzymes have been found exclusively in seeds. Clinical trials are underway for the drug Bragusol, one of the active ingredients of which is a lipase inhibitor from spring rapeseed seeds (Brassica napus var. oleifera). The drug is intended for the treatment of Upper Respiratory Tract infections. The anti-inflammatory property of Bragusol is provided by the lipase inhibitor, which is capable of suppressing the activity of lipolytic enzymes in the area of inflammation development.