BIOCHEMISTRY - V. V. Yemelianov - 2016

SECTION 1. ENZYMES

1.1. General characteristics of Enzymes

Enzymes are specific Proteins that act as catalysts in biological systems.

Being protein in nature, Enzymes exhibit all the Properties of Proteins:

- they possess several levels of macromolecular Organization, as confirmed by X-Ray Diffraction data;

- like soluble proteins, they form colloidal solutions;

- they yield positive Color Reactions for proteins;

- they are amphoteric compounds;

- they are prone to Denaturation under METABOLISM/18.html">The Influence of identical factors: Temperature, pH fluctuations, the action of heavy metal salts, and physical factors (ultrasound, ionizing radiation, etc.):

- like proteins, enzymes break down into Amino Acids upon Hydrolysis.

Unlike other proteins, enzymes possess catalytic activity. While sharing properties inherent to inorganic catalysts, they differ significantly from them in A number of characteristics (Table 1).

Class="center">Table 1. Similarities and differences between the catalytic action of enzymes and inorganic catalysts

The Study of enzymes constitutes an independent branch of science known as enzymology. The term "enzyme" (from the Greek en zyme - in Yeast), much like "ferment" (from the Latin fermentum - leaven), refers to a process associated with gas evolution and Fermentation.

1.2. Localization of Enzymes in the Organism

Based on their localization and functionality, enzymes are divided into three groups:

I - general (universal) enzymes;

II - organ-specific enzymes;

III - organelle-specific enzymes.

General enzymes are found in virtually all Cells, supporting cellular vitality by catalyzing reactions of PROTEIN AND NUCLEIC acid Biosynthesis, The formation of Biomembranes and principal Cell Organelles, and Energy Metabolism. Nevertheless, general enzymes across different Tissues and Organs vary in their activity levels.

Enzymes characteristic solely or predominantly of a specific organ or tissue are termed organ-specific. In the Liver, these include arginase, urokinase, histidase, y-glutamyltransferase, Alanine aminotransferase, and sorbitol dehydrogenase. Alkaline phosphatase is an organ-specific enzyme of the Kidneys and Bone tissue, acid phosphatase is characteristic of the Prostate Gland, and α-amylase and lipase are typical of the Pancreas.

Within The Cell, enzymes are also distributed unevenly. Some enzymes exist in a colloid-dissolved state in the Cytosol, whereas others are embedded within cell organelles (structured state). Different cell organelles possess a specific set of enzymes that determine their Functions:

- cell membrane: alkaline phosphatase, adenylate cyclase, K⁺-Na⁺-ATPase;

- Cytoplasm: enzymes of Glycolysis and the Pentose Phosphate Pathway;

- microsomes: enzymes responsible for hydroxylation;

- Ribosomes: enzymes responsible for Protein Synthesis;

- Lysosomes: hydrolytic enzymes;

- Mitochondria: enzymes of The Tricarboxylic Acid Cycle and Oxidative Phosphorylation;

- Cell Nucleus: enzymes responsible for RNA and DNA Synthesis;

- nucleolus: DNA-dependent RNA polymerase.

Cells contain distinct compartments that differ in their enzyme sets and, consequently, in their metabolism (compartmentalization of metabolism).

The determination of organ- or organelle-specific enzyme activities in plasma or Blood serum is widely used in clinical Diagnostics. Elevated enzyme activity in Blood Plasma (serum) is primarily associated with cytolysis (i.e., increased permeability of Introduction/36.html">Biological Membranes or cell necrosis) and the release of enzymes into the bloodstream. In this case, enzyme activity decreases in the damaged organ while increasing in the blood plasma or serum.

1.3. Structure of enzymes

Both simple and complex enzymes exist in nature. Simple enzymes consist entirely of polypeptide chains and yield exclusively amino acids upon hydrolysis. Most natural enzymes are complex proteins containing, In addition to polypeptide chains, a non-protein component—a coenzyme or cofactor. Various interpretations of these terms exist in the literature. We adhere to the view that a coenzyme is the organic non-protein moiety of a complex enzyme, whereas a cofactor is inorganic.

Cofactors are most frequently Metal Ions, such as iron, copper, zinc, etc. Cofactors are required for the expression of catalytic activity in many enzymes.

Many Coenzymes are derivatives of Water-Soluble Vitamins (Table 2).

Table 2. Vitamin-derived coenzymes

Three groups of coenzymes can be distinguished:

- those involved in oxidation-reduction reactions: (НАД⁺/ НАДН₂, НАДФ⁺/НАДФН₂, ФАД/ФАДН₂, KoQ);

- those involved in The transfer of atomic groups: pyridoxal phosphate (amino group transfer), biotin (carbon dioxide transfer), tetrahydrofolic acid (methyl and other single-carbon group transfer);

- those involved in synthesis, isomerization, and Cleavage reactions: thiamine pyrophosphate (keto acid decarboxylation), coenzyme A (for fatty acid activation).

Some coenzymes (FMN, FAD, pyridoxal phosphate) are stably bound to the enzyme and can be considered part of the Active Site. Other coenzymes (НАД⁺, НАДФ⁺, CoA) bind to the active site under physiological conditions only at the moment of the reaction. In this respect, they resemble enzyme substrates. If a coenzyme undergoes chemical changes during the reaction that are directly opposite to those occurring in the substrate, such coenzymes can be regarded as a second substrate or cosubstrate. Moreover, the transformations of the coenzyme itself sometimes have direct physiological significance, for example, the formation of НАДН₂ in the TCA cycle and its oxidation in the Respiratory Chain.

1.3.1. Active Site of Enzymes

Enzymes are characterized by the presence of specific catalytic centers. The active site is the region of the enzyme molecule that specifically interacts with the substrate and takes direct part in catalysis. As a rule, the active site is located not On the surface of the enzyme molecule, but rather within a narrow hydrophobic cleft (pocket), which favors the generation of a high local Substrate Concentration (Fig. 1). Active sites are formed at the level of tertiary structure. Any factors leading to denaturation—i.e., the disruption of tertiary structure—result in the destruction of the active site architecture and, consequently, the loss of the enzyme's catalytic properties.

Fig. 1. Active site of an enzyme (diagram based on Mahler and Cordes): dark bars represent segments of the enzyme polypeptide chain; R denotes amino acid residues and their sequence numbers (starting from the N-terminus)

from the N-terminus

The active site can conventionally be divided into two regions: the substrate-binding site (contact area), which ensures specific complementary binding of the substrate, and the catalytic center proper, which directly enters into chemical interaction with the substrate. The enzyme active site frequently includes a cofactor-binding region.

Most substrates bind to the active site at multiple points. This ensures that the substrate molecule binds in a uniquely defined orientation, dictating the precise pathway of subsequent chemical transformations.

1.3.2. Allosteric Sites of Enzymes

In addition to the active site, a group of regulatory enzymes may contain one or more allosteric sites (derived from the Greek allos meaning "other" and stemma meaning "space" or "structure"), which are located outside the active site. Modulator molecules (either activators or inhibitors) can bind to the allosteric site to regulate enzyme activity. The binding of a modulator induces a conformational change in the enzyme molecule, thereby altering the configuration of the active site and resulting in either an increase or decrease in enzymatic activity (Fig. 2).

Fig. 2. Schematic representation of an allosteric enzyme consisting of two protomers: S - substrate; M₁ - modifier binding at the active site; M₂ - effector binding at the allosteric site

Allosteric sites are by no means present in every enzyme. Typically, they are found in enzymes with a quaternary structure, which are more readily subjected to significant conformational shifts. An enzyme may possess several distinct allosteric sites. Due to their high selectivity, the enzyme's activity responds differently to various modulators.

1.4. Isoenzymes

Isoenzymes (isozymes) are Multiple Forms of an enzyme that catalyze the same chemical reaction but differ in their Physical and Chemical properties (such as substrate affinity, catalytic reaction rate, electrophoretic mobility, varying sensitivity to inhibitors and activators, thermostability, and optimal pH).

A classic example is Lactate dehydrogenase (LDH), an enzyme that catalyzes the reversible reaction:

This enzyme exists in 5 isoforms, each composed of 4 protomers (subunits) of two types (M and H). LDH isoenzymes differ at The quaternary structure level: LDH₁ - 4H; LDH₂ - 3H1M; LDH₃ - 2H2M; LDH₄ - 1H3M; LDH₅ - 4M. The H- and M-type polypeptide chains have similar molecular weights, but the former are rich in dicarboxylic amino acids, whereas the latter are enriched in diamino acids. Consequently, they carry different net charges and can be separated by Electrophoresis. The isoforms are designated according to their electrophoretic mobility toward the anode (Fig. 3).

Fig. 3. Distribution and relative amounts of LDH isoenzymes in various organs. Extracts were applied to the origin line labeled "Start". Under the given experimental conditions (pH), four LDH isoenzymes migrate toward the anode, while one (LDH₅) migrates toward the cathode

During individual development (ontogeny), the relative proportions of isoenzymes in specific tissues undergo changes. Embryonic tissues are dominated by LDH₄ and LDH₅. Postnatal development brings shifts in isoenzyme profiles across various tissues. In tissues reliant on aerobic metabolism, such as the myocardium and Adrenal Glands, LDH₁ and LDH₂ predominate. Conversely, tissues maintaining Anaerobic Metabolism (such as Skeletal Muscle and the liver) are dominated by LDH₄ and LDH₅.

The existence of multiple isoforms enhances the adaptive capacity of tissues, organs, and the organism as a whole to changing environmental conditions. Determining the plasma LDH isoenzyme profile holds significant clinical and diagnostic value for Differential Diagnosis: myocardial infarction is characterized by elevated levels of LDH₁ and LDH₂, whereas liver diseases are marked by increases in LDH₄ and LDH₅.

1.5. Enzyme Specificity

Unlike inorganic catalysts, enzymes are characterized by a high degree of reaction specificity. The specificity of an enzyme is dictated by the three-dimensional structure of its active site.

An enzyme can exhibit extreme selectivity toward its substrate. For instance, the enzyme urease recognizes only a single substance—urea—catalyzing its breakdown into CO₂ and ammonia:

When an enzyme catalyzes The conversion of only one specific substrate, this is known as absolute substrate specificity. However, such instances are relatively rare.

More commonly, substrate specificity is relative (group specificity). This means the enzyme can accelerate a similar type of reaction across a group of structurally related compounds. For example, Alcohol dehydrogenase catalyzes the oxidation not only of ethanol, but also of other aliphatic alcohols:

Digestive enzymes are prime Examples of group specificity. Pepsin, a protease, cleaves peptide bonds in proteins of both animal and plant origins, despite their diverse amino acid compositions. The primary target of pepsin is the -CO-NH- peptide bond. Lipases, which cleave ester bonds, catalyze the hydrolysis of fats into glycerol and Fatty acids, yet they show no activity toward peptide bonds.

Stereochemical specificity arises from the existence of optically active L- and D-isomers or geometric isomers of chemical compounds. For example, the enzyme fumarase catalyzes the conversion of fumaric acid (the trans-isomer) but is inactive toward maleic acid (the cis-isomer):

Most enzymes involved in amino acid transformations act exclusively on L-isomers, whereas enzymes catalyzing carbohydrate conversions act on D-isomers.

Thus, due to their reaction specificity, enzymes ensure that only certain specific pathways proceed at high rates among the vast array of potential transformations within the microenvironment of cells and the intact organism, thereby regulating the intensity of metabolism.

1.6. Mechanism of enzyme Action

Any enzymatic reaction involves several intermediate steps (Fig. 4):

Fig. 4. MAIN STAGES OF Enzymatic Catalysis

Stage I involves the sorption of the substrate (S) by the substrate-binding site of the enzyme's active center (E), resulting in the formation of an enzyme-substrate complex (E-S). This stage is driven by weak non-covalent interactions and is therefore fully reversible. Conformational changes occurring during sorption enhance the spatial fit between the active center and the substrate.

Stage II entails the Covalent Modification of the substrate within the enzyme-substrate complex, yielding a complex with a chemically altered substrate (E-S*). The catalytic center itself participates in this stage, bringing about the cleavage of certain chemical bonds and the formation of new ones.

Consequently, this step is irreversible, except in genuinely reversible reactions. The requirement for covalent transformations makes Stage II the slowest, rate-limiting step of the entire enzymatic process.

Stage III is the desorption of the finished reaction product (P) from the E-S* complex, accompanied by the release of the enzyme in its original form. Like Stage II, this step is irreversible (except in the catalysis of reversible reactions).

1.7. Fundamentals of Enzyme Kinetics

Kinetics investigates the influence of various factors on reaction velocity. The General Principles of chemical reaction kinetics are equally applicable to enzymatic reactions. The rate of an enzymatic reaction is measured by the decrease in substrate concentration or the increase in product concentration per unit time. It is standard practice to measure the initial reaction velocity to minimize The impact of factors such as declining substrate concentration, reaction reversibility, and the accumulation of product inhibitors. Reaction rates are typically expressed in mol/s or µmol/min.

The time interval during which the reaction velocity equals or closely approximates the initial velocity corresponds to the linear segment of the reaction rate versus time curve (Fig. 5).

Fig. 5. Dependence of enzymatic reaction rate on time. The initial velocity (v₀) increases in proportion to the Enzyme Concentration (E)

In vitro experiments make it possible to study an enzymatic reaction by varying a specific parameter while keeping all others constant.

1.7.1. Effect of Enzyme Concentration on Reaction Rate

The dependence of reaction rate on enzyme concentration is studied by varying The amount of enzyme while maintaining a sufficiently high substrate concentration. Under these conditions, the reaction rate within the interval corresponding to the initial velocity is directly proportional to the enzyme concentration in the reaction mixture (Fig. 6).

Fig. 6. Dependence of enzymatic reaction rate on enzyme concentration [E] at a sufficiently high substrate concentration

Dividing the reaction rate measured along the linear segment by the enzyme concentration in the mixture yields a parameter known as enzyme activity. Enzyme activity is defined as the reaction rate referred to the enzyme concentration. Essentially, it represents the slope of the linear segment relative to the abscissa axis. Because enzymes can exist in various functional states, the parameters "enzyme activity" and "amount of enzyme" may not coincide; enzyme activity characterizes the efficiency of the enzyme rather than its quantity.

To quantify the amount of enzyme, the International System of Units (SI) recommends the unit known as the katal. A katal is the amount of enzyme that converts 1 mol of substrate per 1 s.

For biological systems, this unit is excessively large, so the nanokatal is commonly used. More frequently, the more convenient International Unit (IU) is applied. An IU is defined as the amount of enzyme that converts 1 µmol of substrate per 1 min.

1.7.2. Effect of Substrate Concentration on Reaction Rate

The Effect of substrate concentration can be investigated by measuring the reaction velocity across a series of assays featuring a constant enzyme concentration coupled with increasing substrate concentrations.

The plot of the enzyme-catalyzed reaction rate (v) versus substrate concentration at a fixed enzyme concentration is a hyperbola (Fig. 7).

Fig. 7. Dependence of the enzymatic reaction rate on substrate concentration at a constant enzyme concentration

Initially, the reaction rate is directly proportional to the substrate concentration; with its further increase, the reaction rate gradually reaches a maximum value. This means that all binding sites of the enzyme are occupied (saturated). In this region, the reaction rate is independent of the substrate concentration. Such a curve is referred to as the substrate saturation curve.

The equation describing the substrate saturation curve was proposed by Michaelis and Menten and bears their name (the Michaelis-Menten Equation):

where [S] is the substrate concentration;

Ʋ is the reaction rate at a given substrate concentration;

К_М is the Michaelis constant.

Ʋ_max and К_М are the main kinetic constants characterizing the enzyme in its reaction with a given substrate.

It is easy to calculate that if Ʋ = 1/2 Ʋ_max, then К_М = [S], i.e., К_М is the substrate concentration at which the reaction rate is 1/2 Ʋ_max. The Michaelis constant can be determined graphically. The segment on the abscissa corresponding to a rate equal to half of the maximum represents К_М.

Ʋ_max is the maximum velocity of a given enzymatic reaction, i.e., the limiting value approached by the reaction rate as the substrate concentration increases infinitely.

To simplify the determination of v_max and К_М, the Michaelis-Menten equation can be linearized:

1/Ʋ = К_м + [S]/ Ʋ_max • [S]

1/Ʋ = K_M/ Ʋ_max • [S] + 1/ Ʋ_max.

1/Ʋ = K_M/ Ʋ_max - 1/[S] + 1/ Ʋ_max - the Lineweaver-Burk equation.

The equation describing the Lineweaver-Burk plot is the straight-line equation y = kx + b, where 1/Ʋ_max is the y-intercept, K_M/Ʋ_max is the slope of the line, and the intersection of the line with the abscissa gives the value 1/K_M (Fig. 8). The Lineweaver-Burk plot allows К_М to be determined from a relatively small number of points. This plot is also used in evaluating the action of inhibitors.

Fig. 8. Lineweaver-Burk plot

The values of К_М vary over a wide range: from 10^-6 mol/L for highly active enzymes to 10^-2 mol/L for enzymes with low activity. Estimations of К_М are of practical importance. At substrate concentrations 100 times exceeding K_M, the enzyme will operate at virtually maximum efficiency; therefore, Ʋ_max will reflect the amount of active enzyme present. This circumstance is used to estimate the enzyme content in a preparation. In addition, К_М is an intrinsic characteristic of the enzyme.

1.7.3. Dependence of the enzymatic reaction rate on temperature

Enzymes, being proteinaceous substances, exhibit maximum activity within a limited temperature range (Fig. 9). At temperatures up to 40-50 °C, the reaction rate increases According to the theory of chemical kinetics, i.e., approximately doubling in accordance with the van 't Hoff rule. At higher temperatures, thermal denaturation leads to a decrease in the amount of active enzyme and, ultimately, to the complete cessation of the enzymatic reaction. Thermolability distinguishes enzymes from inorganic catalysts.

Fig. 9. Dependence of the enzymatic reaction rate (Ʋ) on ambient temperature

1.7.4. Dependence of the enzymatic reaction rate on pH

The optimum pH for the action of most enzymes lies within the physiological range of 6.0-8.0. Pepsin is active at pH 1.5 - 2.0, which corresponds to the acidity of gastric juice. Arginase, a specific liver enzyme, is active at 10.0. The effect of environmental pH on the rate of an enzymatic reaction is related to the state and degree of ionization of ionogenic groups in the enzyme and substrate molecules (Fig. 10). This factor determines protein conformation, the state of the active center and the substrate, the Formation of the enzyme-substrate complex, and the catalysis process itself.

Fig. 10. Effect of environmental pH on the rate of an enzymatic reaction: 1 - pepsin; 2 - Lysozyme; 3 - salivary amylase; 4 - arginase

1.8. Inhibition of Enzyme Activity

An important feature of enzymes is their inactivation under the influence of specific inhibitors. Inhibitors are substances that cause partial or complete suppression of enzymatic reactions. The Inhibition of enzymatic activity can be irreversible or reversible, competitive or non-competitive.

Irreversible inhibition is a persistent inactivation of the enzyme resulting from the covalent binding of an inhibitor molecule at the active center or another specific site that alters the enzyme conformation. The dissociation of such stable complexes with the regeneration of the free enzyme is practically impossible. To overcome the effects of such inhibition, the organism must synthesize new enzyme molecules.

Reversible inhibition is characterized by the equilibrium complexation of the inhibitor with the enzyme through non-covalent bonds, As a result of which such complexes are capable of dissociation with the restoration of enzyme activity.

The Classification of inhibitors into Competitive and non-competitive is based on whether their action is diminished (competitive inhibition) or not diminished (non-competitive inhibition) when the substrate concentration is increased.

Competitive Inhibitors are typically substances whose structure resembles that of the substrate. This circumstance allows them to bind to the same active center as the substrate, preventing the interaction of the enzyme with the substrate already at the binding stage. Following binding, the inhibitor may be converted into a product or remain in the active center until dissociation occurs.

Reversible competitive inhibition can be represented by the following scheme:

The degree of Enzyme Inhibition is determined by The ratio of substrate and inhibitor concentrations. A classic example of this type of inhibition is the suppression of succinate dehydrogenase activity by malonate, which displaces succinate from the substrate site and prevents its conversion into fumarate.

In irreversible inhibition, covalent binding of the inhibitor occurs at the active center. An example of irreversible inhibition is the inactivation of the enzyme Triosephosphate isomerase by trichloroacetol phosphate. This inhibitor is a structural analogue of the substrate, dihydroxyacetone phosphate, and irreversibly attaches to the glutamic acid residue in the active center.

1.9. Classification and Nomenclature of Enzymes

The modern classification and nomenclature of enzymes were developed by the Enzyme Commission of the International Union of Biochemistry, established in 1956, and approved at the 5th International Biochemical Congress in Moscow in 1961 (Table 3). This classification and nomenclature remain in effect today, being updated with newly discovered enzymes.

Table 3. International Classification of Enzymes

According to the current classification, all enzymes are divided into six classes based on the type of catalyzed reaction (see Table 3). Each enzyme has its own code.

Given The Diversity of enzymes within each class, they are subdivided into subclasses, which in turn are divided into sub-subclasses.

According to the nomenclature, the name of an enzyme should consist of the name of its substrate and the designation of the type of catalyzed reaction, ending with the suffix -ase. If two substrates participate in the reaction, the names of both are given (separated by a colon). Hence the cumbersomeness of systematic names. Therefore, it was decided to "legalize" The Use of many working names, which are much shorter and more convenient:

1. Oxidoreductases - enzymes that catalyze oxidation-reduction reactions. The following features are characteristic of enzymes in this class:

- they form redox systems;

- they catalyze reactions associated with energy release;

- they are complex enzymes, with NAD, FAD, heme, and KoQ acting as coenzymes.

Scientific (systematic) name: name of the electron donor : name of the electron acceptor and class name.

Working name: where possible, the term dehydrogenase or reductase is used. If oxygen is the electron acceptor, oxidase is used.

Example:

Systematic enzyme name: L-lactate: NAD+-oxidoreductase.

Working name: lactate dehydrogenase.

2. Transferases — a class of enzymes catalyzing the transfer of atomic groups or molecular residues from one substrate to another. Depending on the type of transferred group, the following subclasses are distinguished:

- phosphotransferases (-РО₃Н₂);

- aminotransferases (-NH₂);

- methyltransferases (-СН₃);

- Acyltransferasesetc.

Systematic enzyme name: donor of the transferred group: acceptor of the group: name of the group and class name.

Working name: group donor + transferase or acceptor: group + transferase.

Example:

Systematic enzyme name: aspartate: 2-oxoglutarate aminotransferase. Working name: aspartate aminotransferase.

3. Hydrolases — accelerate hydrolytic cleavage reactions. The subclass indicates The Nature of the cleaved bond:

- esterases (cleave ester bonds);

- glycosidases (cleave glycosidic bonds);

- peptidases (hydrolyze peptide bonds).

Systematic enzyme name: substrate name: cleaved group + class name.

Working name: substrate name with the -ase suffix.

Example:

Systematic enzyme name: acetylcholine acetylhydrolase.

Working name: acetylcholinesterase.

4. Lyases. The class of lyases comprises enzymes that catalyze the cleavage of C-O, C-C, C-N, and other bonds, as well as the non-hydrolytic reversible removal of various groups from substrates. These reactions are accompanied by the formation of a double bond or The addition of groups across a double bond.

Systematic enzyme name: substrate name: eliminated group + class name.

Working name: substrate name + class.

Example:

Systematic name: Pyruvate carboxylase.

Working name: pyruvate decarboxylase.

5. Isomerases. This class includes enzymes that catalyze the interconversion of isomers. Based on the type of reaction catalyzed, five subclasses are distinguished:

- racemases and epimerases (catalyzing the interconversion of optical isomers);

- cis-trans isomerases (catalyzing the interconversion of geometric isomers);

- intramolecular oxidoreductases;

- intramolecular transferases (Mutases);

- intramolecular lyases.

6. Ligases (synthetases). The class of ligases includes enzymes that catalyze the synthesis of organic molecules from two precursor molecules, coupled with The energy released by the cleavage of ATP (or another nucleoside triphosphate). Their systematic names are formed using the format "X : Y ligase", where X and Y denote the starting compounds. An example is Z-glutamate:ammonia ligase (commonly known by its recommended abbreviation "Glutamine Synthetase"), which catalyzes the Synthesis of Glutamine from glutamic acid and ammonia in the presence of ATP.

Review Questions

1. Enzymes: definition and role in living systems. Structure of enzymes: active site, allosteric site, and their functions.

2. Coenzymes and cofactors: chemical nature and functions. The Role of vitamins in the expression of enzyme catalytic activity. Comparative characteristics of enzymes and non-biological catalysts.

3. Mechanism of Enzymatic catalysis.

4. International classification and nomenclature of enzymes: construction principles and Enzyme Classification numbers (codes). Characteristics of enzyme classes (oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases).

5. Regulation of enzyme Activity. Dependence of enzymatic reaction rate on enzyme concentration, substrate concentration, temperature, and medium pH. The equations of

Michaelis-Menten and Lineweaver-Burk, the Michaelis constant, and the substrate saturation curve. Competitive and allosteric modulators.

6. Intracellular Localization of Enzymes. Enzymes of cellular organelles (nucleus, mitochondria, ribosomes, lysosomes, Golgi apparatus, Endoplasmic reticulum, Cytoskeleton, centrosome).

Written Homework

Mandatory

1. Draw the structural formula of the tripeptide H₂N-Val-Cys-Asp-COOH and determine the pH range in which its isoelectric point lies.

2. During the formation of the Tertiary Structure of the Insulin hormone, the following fragments of the polypeptide chain are brought into close spatial proximity:

Leu-Val-Cys-Gly-Glu-Arg, Leu-Glu-Asn-Tyr-Cys-Asp.

What types of bonds between The amino acid side chains can be formed in this case?

3. The scheme shows 5 Chemical Reactions occurring in the body, involving Glutamic Acid and Glutamine.

Write out the reaction equations, indicate the classes of enzymes catalyzing them, and name each enzyme according to the systematic nomenclature.

4. The enzyme dipeptidase from the mucosa of the Small Intestine catalyzes the hydrolysis of the dipeptide glycylglycine. The following table shows the dependence of the enzymatic reaction rate on the substrate concentration in the absence and in the presence of the modulator trasylol:

Task:

a) write the equation for the dipeptide hydrolysis reaction;

b) plot the reaction rate versus substrate concentration for both cases in Michaelis–Menten and Lineweaver–Burk coordinates;

c) determine the maximum reaction rate and the Michaelis constant;

d) what type of enzyme activity modulation takes place in this case?

e) to which part of the enzyme molecule does the modulator bind?

Supplementary

1. The scheme shows 5 chemical reactions occurring in the body, involving glutamic acid and glutamine.

Write out the reaction equations, indicate the classes of enzymes catalyzing them, and name each enzyme according to the systematic nomenclature.

2. The enzyme Phosphofructokinase catalyzes the conversion of fructose-6-phosphate to fructose-1,6-diphosphate. The table shows the dependence of the enzymatic reaction rate on the concentration of fructose-6-phosphate in the absence and in the presence of the modulator citrate (citric acid):

Task:

a) plot the reaction rate versus substrate concentration for both cases in Michaelis–Menten and Lineweaver–Burk coordinates;

b) determine the maximum reaction rate and the Michaelis constant;

c) what type of enzyme activity modulation takes place in this case?

d) to which part of the enzyme molecule does the modulator bind?

Standard test control version on the topic "Enzymes"

Instructions: unless otherwise specified in the test question, select a single correct answer.

1. Choose the name of the enzyme that catalyzes the following reaction:

a) asparagine:ammonia ligase;

b) aspartate aminotransferase;

c) asparagine:ammonia lyase;

d) asparagine:H₂O oxidoreductase;

e) asparagine:ammonia hydrolase.

2. The enzyme lipase catalyzes the hydrolysis of triglycerides in adipose tissue. Under the action of another enzyme, protein kinase, phosphate groups are attached to the lipase molecule, which increases lipase activity. What type of ENZYME ACTIVITY REGULATION is this called:

a) competitive;

b) allosteric;

c) covalent modification;

d) induction-repression.

3. Choose a common property of enzymes and non-biological catalysts:

a) activity is regulated by activators and inhibitors;

b) they function only at low temperatures;

c) they are able to accelerate reactions by up to 10¹⁷-fold;

d) they possess high substrate specificity;

e) they lower the activation energy barrier of the reaction.

4. What role do water-soluble vitamins play in enzymatic catalysis:

a) they are simple enzymes;

b) they are complex enzymes;

c) they serve as coenzyme precursors;

d) they serve as allosteric sites of enzymes;

d) are competitive Enzyme Inhibitors.

5. Which cellular organelles contain the enzymes responsible for DNA Replication and Repair:

a) endoplasmic reticulum;

b) nucleus;

c) ribosomes;

d) Golgi apparatus;

e) lysosomes.

6. Which characteristic of an enzyme remains unchanged upon denaturation:

a) water solubility;

b) biological activity;

c) Michaelis constant;

d) Primary Structure;

e) solution viscosity.

7. The enzyme's active center, containing Serine, glutamic acid, and valine, is most likely to interact with:

8. Which scientist proposed the theory according to which enzymatic catalysis involves the mutual adaptation of the enzyme and substrate in terms of geometric and electronic structure:

a) E. Fischer;

b) L. Michaelis;

c) M. Menten;

d) D. Koshland;

e) A. Cornish-Bowden.

9. Fill in the missing words (3 Answers) in the sentence: "A reversible competitive inhibitor is structurally similar to ..., binds to the ... center of the enzyme, and is displaced from it by an excess of ..."

10. Write the names and numbers of the enzyme classes (6 answers) that catalyze reactions 1, 2, and 3: