LECTURE No. 2

State Budgetary Educational Institution of Higher Professional Education of the USMU of the Ministry of Health of the Russian Federation
Department of Biochemistry
Discipline: Biochemistry
LECTURE No. 2
Enzymes 2.
Lecturer: Gavrilov I.V.
Faculty: therapeutic and prophylactic,
Course: 2
Ekaterinburg, 2015

Lecture outline

1.
2.
3.
Kinetics of enzymatic reactions.
Regulation of the rate of enzymatic reactions.
Cell signaling

Enzymology is the science
studying enzymes

1. Kinetics
enzymatic reactions
Kinetics of enzymatic reactions is a branch of enzymology that studies
influence of reacting substances (substrates,
products, inhibitors, activators, etc.) and
conditions (pH, t°, pressure) on speed
enzymatic reaction.

Theories about the mechanisms of enzyme action

Theories about specificity
enzyme action
1. Key-lock model
To explain the high specificity of enzymes according to
in relation to substrates, Emil Fischer put forward in 1894
hypothesis of strict correspondence of geometric shape
substrate and active site of the enzyme.
+
E+S
ES
E
P1
+
P2

2. The theory of “induced correspondence”
S
A
B
E
A
B
C
C
There is not only
geometric, but also
electrostatic
correspondence
ES
Theory of induced (forced) correspondence
Daniel Koshland (1959): complete enzyme compliance
and substrate occurs only in the process of their interaction:
The substrate induces the necessary conformational
changes in the enzyme, after which they are connected.
The theory is based on kinetic analysis data,
studying enzyme-substrate complexes using methods
X-ray structural analysis, spectrography and
crystallography, etc.

3. The theory of “induced correspondence”
(modern views)
S
A
B
A
C
B
C
E
ES
When an enzyme and substrate interact, both
undergo modifications and adapt to each other
friend. The changes that occur in the substrate contribute to
turning it into a product.

Transition state theory
(intermediate connections)
P
S
E
ES
ES*
EP*
E
when enzyme E interacts with substrate S, it forms
complex ES*, in which the reactivity
substrate is higher than in the native state. Through the row
intermediate compounds are converted
substrate into reaction product P

Mechanisms of enzymatic reactions

During enzymatic catalysis, those
the same mechanisms that are possible without the participation
enzymes:
1.
2.
3.
4.
Acid-base reactions – in the active site
enzyme there are groups -COO- and -NH3+, which
capable of attaching and giving away N.
Addition reactions (elimination, substitution)
electrophilic, nucleophilic - in the active center
enzyme there are heteroatoms displacing
electron density.
Redox reactions – in
the active center of an enzyme contains atoms
having different electronegativity
Radical reactions.

Energy of enzymatic reactions

Enzymes reduce activation energy
The rate of a chemical reaction depends on
concentrations of reactants
Substrates in combination with enzymes
become more resilient
intermediate compounds, due to which they
the concentration increases sharply, which
helps speed up the reaction

Non-enzymatic reaction
S
S*
P*
P
S
E
ES
ES*
Enzymatic reaction
EP*
E

ENERGY BARRIER OF REACTION –
amount of energy needed
molecule to enter into chemical
reaction.
ACTIVATION ENERGY - amount of energy,
which must be communicated to the molecule
to overcome energy
barrier.

Free energy of the system
S*
Activation energy
non-catalyzed reaction
S
ES*
Activation energy
catalyzed reaction
Original
state
P
Final state
Progress of the reaction

2H2O + O2
2.
3.
Energy
activation
1) 2H2O2
Free energy of the system
Catalase
1.
Progress of the reaction
Activation energy:
1. In a spontaneous reaction – 18 kcal/mol
2. When using Fe2+ catalyst – 12 kcal/mol
3. In the presence of the enzyme catalase – 5 kcal/mol

Dependence of reaction rate on substrate concentration

Kinetics
enzymatic reactions
Reaction rate dependence
on substrate concentration
Vmax
Concentration
enzyme constant
[S]

Reaction rate dependence
on enzyme concentration
V
Concentration
substrate –
constant
concentration
enzyme

Effect of temperature on the rate of enzymatic reaction

Temperature increase by 10
degrees increases speed
chemical reaction by 2-4 times.
When the temperature rises, the enzyme
undergoes denaturation and loses
your activity.

Speed
enzymatic
reactions
V
Quantity
active
enzyme
0
10
20
Speed
active reactions
enzyme
30
40
50
60
T

Effect of pH on the rate of enzymatic reaction

Changing the H+ concentration changes
chemical composition of the enzyme, its
structure and catalytic activity.
Changing the H+ concentration changes
chemical composition of the substrate, its
structure and ability to enter into
enzymatic reaction.
Denaturation of the enzyme at very
high or very low pH.

Dependence of the rate of enzymatic reaction on pH

V
0
4
5
6
7
8
9
pH

Michaelis-Menton constant

Km – substrate concentration [S], at which
the rate of enzymatic reaction V is equal to
half the maximum
Vmax
Vmax
2
Km
[S]

Enzyme Reaction Rate Equation

Vmax [S]
V = -----Km + [S]
V – reaction speed
Vmax – maximum reaction speed
Km – Michaelis constant
[S] – substrate concentration

The influence of activators and inhibitors on the rate of enzymatic reactions

Enzymatic inhibition reactions
processes
TYPES OF ENZYME INHIBITION
I. Reversible
II. Irreversible
Competitive
Non-competitive
Non-competitive
Mixed type
Dialysis is performed to determine the reversibility of inhibition.
environment where there is an enzyme and an inhibitor.
If enzyme activity is restored after dialysis, then
inhibition is reversible

Interaction options
inhibitor with enzyme
1. Block the active center of the enzyme
2. Change the quaternary structure of the enzyme
3. Connect with coenzyme, activator
4. Block the part of the enzyme that connects to
coenzyme
5. They disrupt the interaction of the enzyme with
substrate
6. Cause denaturation of the enzyme
(nonspecific inhibitors)
7. Binds to the allosteric center

Competitive type of inhibition
Carried out by a substance similar in chemical
structure to the substrate
V
Vmax
Vmax/2
Km
Kmi
[S]

Non-competitive type of inhibition
The inhibitor reacts with the enzyme in a manner other than
substrate, so increasing the substrate concentration does not
may displace the inhibitor and restore activity
enzyme
V
Vmax
Vmax
Vmax
Vmax
K
m
[S]

2. Regulation of the rate of enzymatic reactions in the body

The most important property of living organisms is the ability to maintain homeostasis. Homeostasis in the body is maintained by regulation

The most important property of living organisms is
ability to maintain homeostasis.
Homeostasis in the body is maintained by
regulation of the rate of enzymatic reactions, which
carried out by changing:
I). Availability of substrate and coenzyme molecules;
II). Catalytic activity of enzyme molecules;
III). Number of enzyme molecules.
E*
S
S
Coenzyme
Vitamin
Cell
P
P

I. Availability of substrate and coenzyme molecules

Transport of substances across the membrane
ATP
ADF + Fn
antiport
Diffusion Facilitated
Diffusion
Cell
Primary active
transport
Secondary active
transport

Insulin
Glucose
GLUT-4
GLUT-4
Adipocytes,
myocytes
E1, E2, E3…
Glucose
PVK
Coenzymes
Hepatocyte
Vitamins
Enzymes
Coenzymes

II. Regulation of enzyme catalytic activity

Regulation of the catalytic activity of enzymes occurs:
1). Non-specific. Catalytic activity of all enzymes
depends on temperature, pH and pressure.
V
pepsin
V
0
50
100
t
0
arginase
7
14
pH
2). Specific. Under the influence of specific activators and
inhibitors, the activity of regulatory enzymes changes,
which control the rate of metabolic processes in
body.

Mechanisms of specific regulation
catalytic activity of enzymes:
1). Allosteric regulation;
2). Regulation by protein-protein
interactions;
3). Regulation through covalent modification.
A). Regulation by
phosphorylation/dephosphorylation
enzyme;
b). Regulation by partial proteolysis.

1. Allosteric regulation

Allosteric enzymes are enzymes whose activity
regulated by reversible non-covalent attachment
modulator (activator and inhibitor) to the allosteric center.
E1
S
E2
A
E3
B
E4
C
P
Activation occurs according to the principle of direct positive
connection, and inhibition is based on the principle of negative feedback
communications.
The activity of allosteric enzymes varies greatly
fast

2. Regulation of the catalytic activity of enzymes using protein-protein interactions

A). Activation of enzymes as a result of the attachment of regulatory proteins.
AC
G
G
AC
ATP cAMP
b). Regulation of enzyme catalytic activity
association/dissociation of protomers
cAMP
cAMP
R
R
C
R
C
PC A
cAMP
S
C
P
R
cAMP
S
C
P

3). Regulation of the catalytic activity of enzymes by their covalent modification

Regulation of enzyme activity is carried out as a result
covalent addition or cleavage of a fragment from it.
There are 2 types:
A). by phosphorylation and dephosphorylation of enzymes; .
ATP
ADF
PC
ENZYME
H3PO4
FPF
*
ENZYME-F
substrate
product
H2O
b). by partial proteolysis of enzymes (extracellular)
Substrate
Trypsinogen
Product
Trypsin

III. Mechanisms for regulating the number of enzymes
Inductors
Repressors
hydrolysis
biosynthesis
Amino acids
Enzyme
Amino acids
Inducers are substances that trigger the synthesis of enzymes
The process of starting enzyme synthesis is called induction
Enzymes, the concentration of which depends on the addition
inducers are called inducible enzymes
Enzymes whose concentration is constant and not regulated
inducers are called constitutive enzymes
Baseline is the concentration of the enzyme being induced
in the absence of an inductor.

Repressors (more precisely corepressors) are substances that
which stop the synthesis of enzymes.
The process of stopping enzyme synthesis is called
repression.
Derepression is a process called
resumption of enzyme synthesis after removal
from the repressor environment
Act as inducers and repressors
some metabolites, hormones and biologically
active substances.

3. Cell signaling

In multicellular organisms, maintenance
homeostasis is provided by 3 systems:
1). Nervous
2). Humoral
3). Immune
Regulatory systems operate with the participation of
signaling molecules.
Signal molecules are organic
substances that carry information.
To transmit a signal:
A). The CNS uses neurotransmitters
B). The humoral system uses hormones
IN). The immune system uses cytokines.

Hormones are signaling molecules of wireless systemic action
True hormones, as opposed to other signaling molecules:
1. synthesized in specialized endocrine cells,
2. transported by blood
3. act remotely on target tissue.
Hormones are divided according to their structure:
1. protein (hormones of the hypothalamus, pituitary gland),
2. amino acid derivatives (thyroid, catecholamines)
3. steroids (sexual, corticoids).
Peptide hormones and catecholamines are soluble in water,
they regulate predominantly the catalytic
enzyme activity.
Steroid and thyroid hormones are water insoluble,
they regulate mainly the amount
enzymes.

Cascade systems
Hormones regulate the amount and catalytic
enzyme activity is not direct, but
indirectly through cascade systems
Hormones
Cascade systems
Enzymes
x 1000000
Cascade systems:
1. They repeatedly enhance the hormone signal (increase the amount or
catalytic activity of the enzyme) so that 1 molecule of hormone
can cause changes in cell metabolism
2. Provide signal penetration into the cell (water-soluble
hormones do not enter the cell on their own)

cascade systems consist of:
1. receptors;
2. regulatory proteins (G-proteins, IRS, Shc, STAT, etc.).
3. secondary intermediaries (messenger - messenger)
(Ca2+, cAMP, cGMP, DAG, ITP);
4. enzymes (adenylate cyclase, phospholipase C,
phosphodiesterase, protein kinases A, C, G,
phosphoprotein phosphatase);
Types of cascade systems:
1. adenylate cyclase,
2. guanylate cyclase,
3. inositol triphosphate,
4. RAS, etc.),

Receptors

Receptors are proteins embedded in the cell membrane or
located inside the cells, which, interacting with
signaling molecules, change the activity of regulatory proteins.
Based on localization, receptors are divided into:
1) cytoplasmic;
2) nuclear;
3) membrane.
Based on their effect, receptors are divided into:
activator (activate cascade systems)
inhibitory (block cascade systems).
According to the mechanism of signal transmission, receptors are divided into 4 types:
1). Ion channel coupled receptors
2). Receptors with enzymatic activity.
There are 3 types:
A). Receptors with tyrosine kinase activity (tyrosine
protein kinase).
b). Receptors with phosphatase activity (tyrosine
protein phosphotase) (for example, PPP).
V). Receptors with guanylate cyclase activity (GC).
3). Receptors coupled with G-proteins in their structure are still
called serpentine.
4). Nuclear and cytoplasmic receptors.

Ion channel coupled receptor

Receptor activity coupled to G protein (serpentine)

Receptor with enzymatic activity (tyrosine kinase)
insulin
a
a
insulin
insulin
a
a
a
b
b
b
b
shooting gallery
shooting gallery
shooting gallery
shooting gallery
ATP
ADF
b
a
b
tier-F* tier-F*
IRS-1
IRS-1-F*
ATP ADP
FPF
FPF*

Adenylate cyclase system
Hormones:
Glucagon, Vasopressin, Catecholamines (via β2-adrenergic receptors)
Pituitary hormones (ACTH, LDH, FSH, LT, MSH, TSH), parathyroid hormone, growth factor
nerves
PGE1
G
R
Cytoplasmic membrane
G
A
C
cytoplasm
ATP cAMP
PC A
Enzyme inactive
PC A*
ATP
ADF
Enzyme act
substrate
F
product
Available
αand
β-adrenergic
receptors
membranes of liver cells, muscles and adipose tissue.
V
plasmatic

Guanylate cyclase system
Signaling molecules:
PNF (relaxation of vascular tone),
Catecholamines (via α-adrenergic receptors)
Bacterial endotoxin (blocks water absorption causing diarrhea)
NO, LPO products (cytoplasmic GC)
G
GC
Cytoplasmic membrane
cytoplasm
GTP cGMP
PC G
Enzyme inactive
PC G*
ATP
ADF
Enzyme act
substrate
F
product
The guanylate cyclase system functions in the lungs, kidneys, and endothelium
intestines, heart, adrenal glands, retina, etc. It is involved in the regulation
water-salt metabolism and vascular tone, causes relaxation, etc.

Inositol triphosphate system
Hormones:
gonadoliberin, thyrotropin-releasing hormone, dopamine, thromboxanes A2, endoperoxides,
leukotrienes, agniotensin II, endothelin, parathyroid hormone, neuropeptide Y,
adrenergic catecholamines (via α1 receptors), acetylcholine,
bradykinin, vasopressin (via V1 receptors).
G
R
G
FL S
Cytoplasmic membrane
FIF2
DG
2+
ITF Ca
substrate
Calmodulin -4Ca2+
PC C
cytoplasm
product
Enzyme inactive
Ca2+
Calmodulin -4Ca2+
Calmodulin
Enzyme act
substrate
product

Transmembrane information transfer involving
cytoplasmic receptors
protein
G
chaperone
Cytoplasmic
membrane
CPR
protein
G
chaperone
Hormones:
Corticoids,
sexual,
thyroid
G
CORE
CPR
G
CPR
DNA
cytoplasm
substrate
product
Transcription
mRNA
Broadcast
mRNA
Enzyme
ribosome

ChapterIV.3.

Enzymes

Metabolism in the body can be defined as the totality of all chemical transformations to which compounds coming from outside undergo. These transformations include all known types of chemical reactions: intermolecular transfer of functional groups, hydrolytic and non-hydrolytic cleavage of chemical bonds, intramolecular rearrangement, new formation of chemical bonds and redox reactions. Such reactions occur in the body at extremely high speed only in the presence of catalysts. All biological catalysts are substances of protein nature and are called enzymes (hereinafter F) or enzymes (E).

Enzymes are not components of reactions, but only accelerate the achievement of equilibrium by increasing the rate of both direct and reverse conversion. Acceleration of the reaction occurs due to a decrease in the activation energy - the energy barrier that separates one state of the system (the initial chemical compound) from another (the reaction product).

Enzymes speed up a variety of reactions in the body. So, quite simple from the point of view of traditional chemistry, the reaction of the elimination of water from carbonic acid with the formation of CO 2 requires the participation of an enzyme, because without it, it proceeds too slowly to regulate blood pH. Thanks to the catalytic action of enzymes in the body, it becomes possible for reactions to occur that without a catalyst would proceed hundreds and thousands of times slower.

Properties of enzymes

1. Influence on the rate of a chemical reaction: enzymes increase the rate of a chemical reaction, but are not consumed themselves.

The rate of a reaction is the change in the concentration of reaction components per unit time. If it goes in the forward direction, then it is proportional to the concentration of the reactants, if in the opposite direction, then it is proportional to the concentration of the reaction products. The ratio of the rates of forward and reverse reactions is called the equilibrium constant. Enzymes cannot change the values ​​of the equilibrium constant, but the state of equilibrium occurs faster in the presence of enzymes.

2. Specificity of enzyme action. 2-3 thousand reactions take place in the cells of the body, each of which is catalyzed by a specific enzyme. The specificity of an enzyme's action is the ability to accelerate the course of one specific reaction without affecting the speed of others, even very similar ones.

There are:

Absolute– when F catalyzes only one specific reaction ( arginase– breakdown of arginine)

Relative(group special) – F catalyzes a certain class of reactions (for example, hydrolytic cleavage) or reactions involving a certain class of substances.

The specificity of enzymes is due to their unique amino acid sequence, which determines the conformation of the active center that interacts with the reaction components.

A substance whose chemical transformation is catalyzed by an enzyme is called substrate ( S ) .

3. Enzyme activity – the ability to accelerate the reaction rate to varying degrees. Activity is expressed in:

1) International units of activity - (IU) the amount of enzyme that catalyzes the conversion of 1 µM of substrate in 1 minute.

2) Catalach (kat) - the amount of catalyst (enzyme) capable of converting 1 mole of substrate in 1 s.

3) Specific activity - the number of activity units (any of the above) in the test sample to the total mass of protein in this sample.

4) Less commonly used is molar activity - the number of substrate molecules converted by one enzyme molecule per minute.

Activity depends primarily on temperature . This or that enzyme exhibits its greatest activity at the optimal temperature. For F of a living organism, this value is in the range +37.0 - +39.0° C, depending on the type of animal. As the temperature decreases, the Brownian motion slows down, the diffusion rate decreases and, consequently, the process of complex formation between the enzyme and the reaction components (substrates) slows down. If the temperature rises above +40 - +50° The enzyme molecule, which is a protein, undergoes a process of denaturation. In this case, the rate of the chemical reaction noticeably drops (Fig. 4.3.1.).

Enzyme activity also depends on pH of the environment . For most of them, there is a certain optimal pH value at which their activity is maximum. Since a cell contains hundreds of enzymes and each of them has its own pH limits, pH changes are one of the important factors in the regulation of enzymatic activity. So, as a result of one chemical reaction with the participation of a certain enzyme, the pH value of which lies in the range of 7.0 - 7.2, a product is formed that is an acid. In this case, the pH value shifts to the region of 5.5 – 6.0. The activity of the enzyme decreases sharply, the rate of product formation slows down, but at the same time another enzyme is activated, for which these pH values ​​are optimal and the product of the first reaction undergoes further chemical transformation. (Another example about pepsin and trypsin).

Chemical nature of enzymes. The structure of the enzyme. Active and allosteric centers

All enzymes are proteins with a molecular weight from 15,000 to several million Da. According to their chemical structure they are distinguished simple enzymes (consisting only of AA) and complex enzymes (have a non-protein part or a prosthetic group). The protein part is called - apoenzyme, and non-protein, if it is covalently linked to the apoenzyme, it is called coenzyme, and if the bond is non-covalent (ionic, hydrogen) – cofactor . The functions of the prosthetic group are as follows: participation in the act of catalysis, contact between the enzyme and the substrate, stabilization of the enzyme molecule in space.

The role of cofactor is usually played by inorganic substances - ions of zinc, copper, potassium, magnesium, calcium, iron, molybdenum.

Coenzymes can be considered as an integral part of the enzyme molecule. These are organic substances, among which there are: nucleotides ( ATP, UMF, etc.), vitamins or their derivatives ( TDF– from thiamine ( IN 1), FMN– from riboflavin ( AT 2), coenzyme A– from pantothenic acid ( AT 3), NAD, etc.) and tetrapyrrole coenzymes - hemes.

In the process of catalyzing a reaction, not the entire enzyme molecule comes into contact with the substrate, but a certain part of it, which is called active center. This zone of the molecule does not consist of a sequence of amino acids, but is formed by twisting the protein molecule into a tertiary structure. Individual sections of amino acids come closer to each other, forming a specific configuration of the active center. An important feature of the structure of the active center is that its surface is complementary to the surface of the substrate, i.e. AK residues in this zone of the enzyme are capable of entering into chemical interactions with certain groups of the substrate. One can imagine that The active site of the enzyme coincides with the structure of the substrate like a key and a lock.

IN active center two zones are distinguished: binding center, responsible for substrate attachment, and catalytic center, responsible for the chemical transformation of the substrate. The catalytic center of most enzymes includes AAs such as Ser, Cys, His, Tyr, Lys. Complex enzymes have a cofactor or coenzyme at the catalytic center.

In addition to the active center, a number of enzymes are equipped with a regulatory (allosteric) center. Substances that affect its catalytic activity interact with this zone of the enzyme.

Mechanism of action of enzymes

The act of catalysis consists of three successive stages.

1. Formation of an enzyme-substrate complex upon interaction through the active center.

2. Binding of the substrate occurs at several points in the active center, which leads to a change in the structure of the substrate and its deformation due to changes in the bond energy in the molecule. This is the second stage and is called substrate activation. In this case, a certain chemical modification of the substrate occurs and it is converted into a new product or products.

3. As a result of this transformation, the new substance (product) loses its ability to be retained in the active center of the enzyme and the enzyme-substrate, or rather, enzyme-product complex dissociates (breaks up).

Types of catalytic reactions:

A+E = AE = BE = E + B

A+B +E = AE+B = ABE = AB + E

AB+E = ABE = A+B+E, where E is the enzyme, A and B are substrates or reaction products.

Enzymatic effectors - substances that change the rate of enzymatic catalysis and thereby regulate metabolism. Among them there are inhibitors - slow down the reaction rate and activators - accelerating the enzymatic reaction.

Depending on the mechanism of reaction inhibition, competitive and non-competitive inhibitors are distinguished. The structure of the competitive inhibitor molecule is similar to the structure of the substrate and coincides with the surface of the active center like a key and a lock (or almost coincides). The degree of this similarity may even be higher than with the substrate.

If A+E = AE = BE = E + B, then I+E = IE¹

The concentration of the enzyme capable of catalysis decreases and the rate of formation of reaction products drops sharply (Fig. 4.3.2.).

A large number of chemical substances of endogenous and exogenous origin (i.e., those formed in the body and coming from outside - xenobiotics, respectively) act as competitive inhibitors. Endogenous substances are regulators of metabolism and are called antimetabolites. Many of them are used in the treatment of oncological and microbial diseases, as. they inhibit key metabolic reactions of microorganisms (sulfonamides) and tumor cells. But with an excess of substrate and a low concentration of the competitive inhibitor, its effect is canceled.

The second type of inhibitors is non-competitive. They interact with the enzyme outside the active site and excess substrate does not affect their inhibitory ability, as is the case with competitive inhibitors. These inhibitors interact either with certain groups of the enzyme (heavy metals bind to the thiol groups of Cys) or most often with the regulatory center, which reduces the binding ability of the active center. The actual process of inhibition is the complete or partial suppression of enzyme activity while maintaining its primary and spatial structure.

A distinction is also made between reversible and irreversible inhibition. Irreversible inhibitors inactivate the enzyme by forming a chemical bond with its AK or other structural components. This is usually a covalent bond to one of the active site sites. Such a complex practically does not dissociate under physiological conditions. In another case, the inhibitor disrupts the conformational structure of the enzyme molecule and causes its denaturation.

The effect of reversible inhibitors can be removed when there is an excess of substrate or under the influence of substances that change the chemical structure of the inhibitor. Competitive and non-competitive inhibitors are in most cases reversible.

In addition to inhibitors, activators of enzymatic catalysis are also known. They:

1) protect the enzyme molecule from inactivating influences,

2) form a complex with the substrate that binds more actively to the active center of F,

3) interacting with an enzyme that has a quaternary structure, they separate its subunits and thereby open up access for the substrate to the active center.

Distribution of enzymes in the body

Enzymes involved in the synthesis of proteins, nucleic acids and energy metabolism enzymes are present in all cells of the body. But cells that perform special functions also contain special enzymes. Thus, the cells of the islets of Langerhans in the pancreas contain enzymes that catalyze the synthesis of the hormones insulin and glucagon. Enzymes that are characteristic only of the cells of certain organs are called organ-specific: arginase and urokinase- liver, acid phosphatase- prostate. By changing the concentration of such enzymes in the blood, the presence of pathologies in these organs is judged.

In a cell, individual enzymes are distributed throughout the cytoplasm, others are embedded in the membranes of mitochondria and the endoplasmic reticulum, such enzymes form compartments, in which certain, closely interconnected stages of metabolism occur.

Many enzymes are formed in cells and secreted into anatomical cavities in an inactive state - these are proenzymes. Proteolytic enzymes (that break down proteins) are often formed as proenzymes. Then, under the influence of pH or other enzymes and substrates, their chemical modification occurs and the active center becomes accessible to the substrates.

There are also isoenzymes - enzymes that differ in molecular structure, but perform the same function.

Nomenclature and classification of enzymes

The name of the enzyme is formed from the following parts:

1. name of the substrate with which it interacts

2. nature of the catalyzed reaction

3. name of the enzyme class (but this is optional)

4. suffix -aza-

pyruvate - decarboxyl - aza, succinate - dehydrogen - aza

Since about 3 thousand enzymes are already known, they need to be classified. Currently, an international classification of enzymes has been adopted, which is based on the type of reaction catalyzed. There are 6 classes, which in turn are divided into a number of subclasses (presented only selectively in this book):

1. Oxidoreductases. Catalyze redox reactions. They are divided into 17 subclasses. All enzymes contain a non-protein part in the form of heme or derivatives of vitamins B2, B5. The substrate undergoing oxidation acts as a hydrogen donor.

1.1. Dehydrogenases remove hydrogen from one substrate and transfer it to other substrates. Coenzymes NAD, NADP, FAD, FMN. They accept the hydrogen removed by the enzyme, transforming it into a reduced form (NADH, NADPH, FADH) and transfer it to another enzyme-substrate complex, where they release it.

1.2. Oxidases - catalyze the transfer of hydrogen to oxygen to form water or H 2 O 2. F. Cytochrome oxidase respiratory chain.

RH + NAD H + O 2 = ROH + NAD + H 2 O

1.3. Monoxidases - cytochrome P450. According to its structure, it is both a hemoprotein and a flavoprotein. It hydroxylates lipophilic xenobiotics (according to the mechanism described above).

1.4. PeroxidasesAnd catalase- catalyze the decomposition of hydrogen peroxide, which is formed during metabolic reactions.

1.5. Oxygenases - catalyze reactions of oxygen addition to the substrate.

2. Transferases - catalyze the transfer of various radicals from a donor molecule to an acceptor molecule.

A A+ E + B = E A+ A + B = E + B A+ A

2.1. Methyltransferase (CH 3 -).

2.2.Carboxyl- and carbamoyltransferases.

2.2. Acyltransferases – Coenzyme A (transfer of acyl group - R -C=O).

Example: synthesis of the neurotransmitter acetylcholine (see chapter “Protein Metabolism”).

2.3. Hexosyltransferases catalyze the transfer of glycosyl residues.

Example: the cleavage of a glucose molecule from glycogen under the influence of phosphorylases.

2.4. Aminotransferases - transfer of amino groups

R 1- CO - R 2 + R 1 - CH - N.H. 3 - R 2 = R 1 - CH - N.H. 3 - R 2 + R 1- CO - R 2

They play an important role in the transformation of AK. The common coenzyme is pyridoxal phosphate.

Example: alanine aminotransferase(ALAT): pyruvate + glutamate = alanine + alpha-ketoglutarate (see chapter “Protein Metabolism”).

2.5. Phosphotransferase (kinase) - catalyze the transfer of a phosphoric acid residue. In most cases, the phosphate donor is ATP. Enzymes of this class mainly take part in the breakdown of glucose.

Example: Hexo(gluco)kinase.

3. Hydrolases - catalyze hydrolysis reactions, i.e. splitting of substances with addition at the site where the water bond is broken. This class includes mainly digestive enzymes; they are single-component (do not contain a non-protein part)

R1-R2 +H 2 O = R1H + R2OH

3.1. Esterases - break down ester bonds. This is a large subclass of enzymes that catalyze the hydrolysis of thiol esters and phosphoesters.
Example: NH 2 ).

Example: arginase(urea cycle).

4.Lyases - catalyze reactions of molecular splitting without adding water. These enzymes have a non-protein part in the form of thiamine pyrophosphate (B 1) and pyridoxal phosphate (B 6).

4.1. C-C bond lyases. They are usually called decarboxylases.

Example: pyruvate decarboxylase.

5.Isomerases - catalyze isomerization reactions.

Example: phosphopentose isomerase, pentose phosphate isomerase(enzymes of the non-oxidative branch of the pentose phosphate pathway).

6.Ligases catalyze reactions for the synthesis of more complex substances from simpler ones. Such reactions require the energy of ATP. “Synthetase” is added to the name of such enzymes.

REFERENCES FOR THE CHAPTER IV.3.

1. Byshevsky A. Sh., Tersenov O. A. Biochemistry for the doctor // Ekaterinburg: Uralsky Rabochiy, 1994, 384 pp.;

2. Knorre D. G., Myzina S. D. Biological chemistry. – M.: Higher. school 1998, 479 pp.;

3. Filippovich Yu. B., Egorova T. A., Sevastyanova G. A. Workshop on general biochemistry // M.: Enlightenment, 1982, 311 pp.;

4. Leninger A. Biochemistry. Molecular basis of cell structure and functions // M.: Mir, 1974, 956 pp.;

5. Pustovalova L.M. Workshop on biochemistry // Rostov-on-Don: Phoenix, 1999, 540 p.

Description of the presentation LECTURE No. 1 Introduction to biochemistry. Enzymes on slides

Lecture outline I. Biochemistry as a science. Subject, goals and objectives of biochemistry. II. Metabolism. Basic concepts. Types of metabolic reactions. III. Enzymology. 1. Enzymes. Definition, chemical nature, physicochemical properties, biological significance. 2. Comparison of enzymes and inorganic catalysts 3. Structure of enzymes

Biochemistry is a science that studies the substances that make up living organisms, their transformations, as well as the relationship of these transformations with the activity of organs and tissues. Biochemistry is a young science; about a hundred years ago it arose at the intersection of physiology and organic chemistry. The term biochemistry was introduced in 1903 by the German biochemist Carl Neuberg (1877 -1956). I. BIOCHEMISTRY

Biochemistry as a science is divided into: Static (bioorganic chemistry) analyzes the structure and chemical composition of organisms Dynamic studies the metabolism and energy in the body Functional studies the interaction of chemical processes with biological and physiological functions OH H O H O HO HH HO HCO 2 + H 2 O ÀÄÔ + Ôí ÀÒÔ À Ò Ô À Ä Ô + Ô í

According to the objects of research, biochemistry is divided into: biochemistry of humans and animals; plant biochemistry; biochemistry of microorganisms; biochemistry of fungi; biochemistry of viruses. You and I will be engaged in medical biochemistry, one of the branches of biochemistry of humans and animals

The object of medical biochemistry is a person. The purpose of the medical biochemistry course is to study: the molecular foundations of human physiological functions; molecular mechanisms of disease pathogenesis; biochemical basis for the prevention and treatment of diseases; biochemical methods for diagnosing diseases and monitoring the effectiveness of treatment (clinical biochemistry) Objectives of the medical biochemistry course: study theoretical material; gain practical skills in biochemical research; learn to interpret the results of biochemical studies

II. Metabolism The life activity of any organism is based on chemical processes. Metabolism (metabolism) is the totality of all reactions occurring in a living organism A FB C DEEnergy Heat Catabolism Anabolism

Metabolites - substances involved in metabolic processes (substrates, A, B, C, products) Substrate - a substance that enters into a chemical reaction Product - a substance that is formed during a chemical reaction Substrate Product. The sequence of reactions as a result of which the substrate is converted into a product is called the A B metabolic pathway. Organic compounds have a complex structure and are synthesized only during several sequential reactions. Example of a metabolic pathway: Glycolysis, oxidative phosphorylation chain

Substrate Product 2 The sequence of reactions that bypass the main metabolic pathway is called a metabolic shunt A B D EProduct 3 Product 1 Examples of metabolic shunts: 1. pentose phosphate shunt, 2. 2, 3-diphosphoglycerate shunt

S 1 The sequence of reactions during which the resulting product is also the substrate of these reactions is called the metabolic cycle S 2 (P) A CBProduct 1 Product 2 Examples of metabolic cycles: 1. Krebs cycle, 2. Ornithine cycle 3. β cycle - fatty oxidation acids 4. Glucose-lactate cycle, 5. Glucose-alanine cycle

Enzymology is a science, a branch of biochemistry, about enzymes. III. Enzymology, structure and properties of enzymes; enzymatic reactions and mechanisms of their catalysis; regulation of enzyme activity. The subject of enzymology is enzymes. Enzymology studies: Medical enzymology - studies the use of enzymes in medicine.

Almost all reactions in a living organism occur with the participation of enzymes. Enzymes are biological catalysts of a protein nature. The biological role of enzymes is that they catalyze the controlled course of all metabolic processes in the body. Physico-chemical properties Being substances of protein nature, enzymes have all the properties of proteins Definition and chemical nature By 2013, more than 5,000 different enzymes have been described

Features of the action of enzymes 1. They accelerate only thermodynamically possible reactions 2. They do not change the state of equilibrium of reactions, but only accelerate its achievement 3. reactions are accelerated significantly, by 10 8 -10 14 times 4. They act in small quantities 5. They are not consumed in reactions 6. Sensitive to activators and inhibitors. 7. The activity of enzymes is regulated by specific and nonspecific factors 8. Enzymes act only in mild conditions (t = 36 -37ºС, pH ~ 7.4, atmospheric pressure) 9. They have a wide range of action, catalyze most reactions in the body 10. For Enzymes are characterized by high specificity: substrate specificity: ▪ absolute (1 enzyme - 1 substrate), ▪ group (1 enzyme - several similar substrates), ▪ stereospecificity (enzymes work with substrates L or D). catalytic specificity (enzymes catalyze reactions of one of the types of chemical reactions) Common to inorganic and catalysts

1. The active center is a part of the enzyme molecule that specifically interacts with the substrate and is directly involved in catalysis b). Catalytic center. The active center, as a rule, is located in a niche (pocket) Contains at least three points for binding the substrate, due to which the substrate molecule attaches to the active center in the only possible way, which ensures the substrate specificity of the enzyme 1. Active center a). Substrate site (contact area) The structural feature of the catalytic center allows the enzyme to catalyze a reaction using a specific catalysis mechanism: acid-base, electrophilic, nucleophilic, etc. Thus. the catalytic center ensures the choice of the chemical transformation path and the catalytic specificity of the enzyme. Structure of enzymes Enzymes are globular proteins containing an active center

Enzyme t +- 0 Substrate Enzymes are characterized by the presence of specific centers of catalysis Substrate site Catalyte. center Active center + 0 -Product t

02. Alosteric center The group of regulatory enzymes has allosteric centers that are located outside the active center. “+” modulators (activators) can be attached to the allosteric center, increasing the activity of the enzymes. The alosteric center and the contact pad are arranged in a similar way + -0+ Activator

02. Allosteric center Also, “-” modulators (inhibitors) can be attached to the allosteric center, inhibiting the activity of enzymes. -0+ Inhibitor —

According to their composition, enzymes are divided into: Simple Consist of only amino acids - Complex Enzymes consist of: 1. Amino acids; 2. Metal ions 3. Organic substances of non-protein nature 0+ Apoenzyme. Prosthetic group + - 0 The protein part (from amino acids) of a complex enzyme is called Apoenzyme The non-protein part of a complex enzyme is called Prosthetic group Metal ions (cofactors) Organic substances of non-protein nature (coenzymes)

Coenzymes are organic substances of a non-protein nature that participate in catalysis as part of the catalytic site of the active center of the enzyme. The catalytically active form of a complex protein is called a holoenzyme. Holoenzyme = Apoenzyme + Coenzyme. Cofactors - metal ions necessary for the manifestation of the catalytic activity of enzymes are called

The following function as coenzymes: Vitamins Activation Coenzymes PP (nicotinic acid) NAD + , NADP + B 1 (thiamine) B 2 (riboflavin) Thiamine pyrophosphate FAD, FMN B 6 (pyridoxal) Pyridoxal phosphate B 12 Cobalamins Hemes (cytochrome coenzymes); Nucleotides (ribosomal coenzymes); coenzyme Q; FAFS (transferase coenzymes); SAM ; Glutathione (glutathione peroxidase coenzyme); Derivatives of water-soluble vitamins:

— 0+ +- 0++ — 0+ + — 0 Cosubstrate is a prosthetic group that is attached to the protein part by weak non-covalent bonds. A cosubstrate is added to the enzyme at the time of reaction: For example, NAD +, NADP +. +- 0+ Product Enzyme + Substrate Enzyme Cosubstrate Enzyme-substrate complex Cosubstrate- 0+ The prosthetic group is usually tightly associated with the apoenzyme.

Cofactors The ions of potassium, magnesium, calcium, zinc, copper, iron, etc. act as cofactors. They stabilize the substrate molecules and ensure its binding; stabilize the active center of the enzyme, stabilize the tertiary and quaternary structure of the enzyme; provide catalysis. The role of cofactors is varied, they are:

For example, ATP joins kinases only together with Mg 2+ + Substrate (ATP)Cofactor (Mg 2+) + - 0 Enzyme Active substrate (ATP- Mg 2+) - 0+ + - 0+ Enzyme-substrate complex Product (ADP ) — 0+ Enzyme. Cofactor (Mg 2+)

Localization and compartmentalization of enzymes in cells and tissues According to localization in the body, enzymes are divided into: General enzymes (universal) Organ-specific enzymes Organelle-specific enzymes. Organelle-nonspecific enzymes. According to their location in the cell, enzymes are divided into: Creatine kinases, aminotransferases, etc. Glycolysis enzymes, ribosomes, etc.

Found in almost all cells, they provide the basic processes of cell life: 1. General enzymes (universal) Enzymes: glycolysis, Krebs cycle, oxidative phosphorylation, PPS, etc. Synthesis and use of ATP; metabolism of proteins, nucleic acids, lipids, carbohydrates and other organic substances; creation of electrochemical potential; movement, etc.

2. Organ-specific enzymes Bone tissue Alkaline phosphatase Myocardium AST, ALT, CK MB, LDH 1, 2 Kidney Transamidinase a, alkaline phosphatase Liver Arginase, ALT, AST, LDH 4, 5, alkaline phosphatase, γ-glutamyl transpeptidase, glutamate dehydrogenase cholinesterase. Characteristic of certain organs or tissues (or a group of organs and tissues). Ensure that they perform specific functions Prostate Acid phosphatase. Pancreas α-amylase, lipase, γ-glutamyl transpeptidase

Distribution of enzymes in the organs of the liver myoc. Skel. musk Kidney Er Bone Prostate AST ALT LDH CK ALP CP 0 -10% 10 -50% 50 -75% 75 -100%

3. Organelle-specific enzymes Cell membrane Alkaline phosphatase, Adenylate cyclase, K-Na-ATPase Cytoplasm Enzymes of glycolysis, PFS Smooth ER Enzymes of microsomal oxidation Ribosomes Enzymes of protein biosynthesis. Lysosomes Acid phosphatase. Mitochondria Enzymes of oxidative phosphorylation, TCA cycle, β-oxidation of fatty acids Core RNA polymerase, NAD synthetase

Isoenzymes are multiple forms of one enzyme that catalyze the same reaction and differ in chemical composition. Isoenzymes differ in: affinity for the substrate (different Km), maximum speed of the catalyzed reaction, electrophoretic mobility, different sensitivity to inhibitors and activators, optimum p. H thermostability Isoenzymes have a quaternary structure, which is formed by an even number of subunits (2, 4, 6, etc.): Isoenzymes Proteins with a quaternary structure and different subunits create a greater variety of forms due to a smaller number of genes.

Lactate dehydrogenase (LDH) LDH consists of 4 subunits of 2 types M (muscle) and H (heart), which in different combinations form 5 isoforms M (muscle)H (heart) Dicarboxylic AAs predominate in the composition Diaminomonocarboxylic AAs predominate in the composition Ë Ä ÃC O O H C C H 3 O Ï Ê 2 Í À Ä + 2 Í À Ä Í 2 C O O H C C H 3 O H Ë à ê ò à òH enzyme of glycolysis and gluconeogenesis

LDH 1 NNNN LDH 2 NNNM LDH 3 NNMM LDH 4 NMMM LDH 5 MMMM O 2 H (heart) M (muscle) pulmonary epithelium alveolmyocardium, erythrocytes, renal cortex cross-sectional skeletal muscles, hepatocytes. N neutral r. H acidic

Creatine kinase (creatine phosphokinase) CPK consists of 2 subunits of 2 types M (from English, muscle - muscle) and B (from English, brain - brain), which in different combinations form 3 isoforms: CPK BB CPK MMKPK plays an important role in energy exchange of muscle and nerve tissue

Determination of the activity of organelle-specific enzymes and isoenzymes in the blood is widely used in clinical diagnostics: Myocardial infarction AST, ALT, CK MB, LDH 1, 2 Pancreatitis Pancreatic amylase, γ-glutamyl transpeptidase, lipase Hepatitis ALT, AST, LDH 4, 5, γ - glutamyl transpeptidase, glutamate dehydrogenase

Nomenclature - names of individual compounds, their groups, classes, as well as rules for compiling these names Classification - division of something according to selected characteristics Nomenclature and classification of enzymes

The modern nomenclature of enzymes is international, translated into different languages ​​Historical names: (pepsin, trypsin) working names: substrate + aza ending (sucrase) substrate + its chemical. conversion + aza (pyruvate carboxylase) Trivial Systematic The name can accurately identify the enzyme and its catalyzed reaction. Each class is built according to a specific scheme Adopted in 1961 by the International Union of Biochemists

Classification of enzymes Based on the 6 known types of chemical reactions, the enzymes that catalyze them are divided into 6 classes. Based on substrates, transferable groups, etc., several subclasses and subsubclasses are distinguished in each class (from 5 to 23); Each enzyme has its own code CF 1. 1. The first digit indicates the class, the second - the subclass, the third - the subsubclass, the fourth - the serial number of the enzyme in its subclass (in order of discovery). http://www. chem. qmul. ac. uk/iubmb/enzyme/

No. Type of reaction Class Subclass Subclass 1 ORV Oxidoreductases 23 subclasses Oxidases Aerobic DG Anaerobic DG Oxygenases Hydroxyperoxidases 2 transfer of functional groups Transferases 10 subclasses Kinases Aminotransferases Protein kinases Hexokinases 3 Hydrolytic removal of a group from the substrate Hydrolases 13 subclasses Phosphotases FPP 4 Non-hydrolytic removal of a group from the substrate a Lyases 7 subclasses 5 isomerization of isomerase 5 subclasses 6 synthesis due to the energy of high-energy compounds ligase 6 subclasses C-O-ligase, C-S-ligase, C-N-ligase, C-C-ligase

Nomenclature of enzymes There is no single approach to the rules for naming enzymes - each class has its own rules. The name of an enzyme consists of 2 parts: 1 part - the name of the substrate (substrates), 2 part - the type of reaction catalyzed. Ending – AZA; Additional information, if necessary, is written at the end and enclosed in brackets: L -malate + NADP + ↔ PVK + CO 2 + NADH 2 L -malate: NADP + - oxidoreductase (decarboxylating);

1. Oxidoreductases Class name: donor: acceptor (cosubstrate) oxidoreductase R - CH 2 - O H + NAD + R - CH =O + NAD H 2 Systematic name: Alcohol: NAD + oxidoreductase Trivial name: alcohol dehydrogenase Code: CF 1. 1 ℮ - and N +

2. Transferases Name of the class: from where: where to what position – what – transferase donor: acceptor – transported group – transferase AT F + D-hexose ADP + D-hexose -6 f Systematic name: AT F: D -hexose -6 - phosphotransferase Trivial name: hexokinase Code: CF 2. 7. 1. 1 Atoms and molecular residues

3. Hydrolases Name of the class: Substrate - what is cleaved off - hydrolase Substrate - hydrolase Acetylcholine + H 2 O Acetate + Choline Systematic name: Acetylcholine -acyl hydrolase Trivial name: Acetylcholinesterase Code: EC 3. 1. 1.

4. Lyases Class name: substrate: what is cleaved off – lyase L-malate H 2 O + fumarate Systematic name: L-malate: hydro-lyase Trivial name: fumarase Code: EF 4. 2. 1.

5. Isomerases Class name: Substrate – type of isomerization – isomerase Substrate – product – isomerase Fumaric acid Maleic acid Systematic name: Fumarate – cis, trans – isomerase

6. Ligases (synthetases) Class name: substrate: substrate – ligase (energy source) L-glutamate + NH 4 + + ATP L-glutamine + ADP + Fn Systematic name: L-glutamate: ammonia – ligase (ATP → ADP + Fn ) Trivial name: glutamine synthetase Code: KF 6. 3. 1.

Lecture 15. Enzymes: structure, properties, functions.

Lecture outline:

1. General characteristics of enzymes.

2. The structure of enzymes.

3. Mechanism of enzymatic catalysis.

4. Properties of enzymes.

5. Nomenclature of enzymes.

6. Classification of enzymes.

7. isozymes

8. Kinetics of enzymatic reactions.

9. Units of measurement of enzymatic activity

1. General characteristics of enzymes.

Under normal physiological conditions, biochemical reactions in the body proceed at high speeds, which is ensured by biological catalysts of a protein nature - enzymes.

They are studied by the science of enzymology - the science of enzymes (enzymes), specific proteins - catalysts synthesized by any living cell and activating various biochemical reactions occurring in the body. Some cells can contain up to 1000 different enzymes.

2. The structure of enzymes.

Enzymes are proteins with high molecular weight. Like any proteins, enzymes have primary, secondary, tertiary and quaternary levels of molecular organization. Primary structure is a sequential combination of amino acids and is determined by the hereditary characteristics of the body; it is it that largely characterizes the individual properties of enzymes. Secondary structure enzymes are organized in the form of an alpha helix. Tertiary structure has the form of a globule and participates in the formation of active and other centers. Many enzymes have quaternary structure and represent a union of several subunits, each of which is characterized by three levels of organization of molecules that differ from each other, both in qualitative and quantitative terms.

If enzymes are represented by simple proteins, that is, they consist only of amino acids, they are called simple enzymes. Simple enzymes include pepsin, amylase, lipase (almost all gastrointestinal enzymes).

Complex enzymes consist of protein and non-protein parts. The protein part of the enzyme is called - apoenzyme, non-protein – coenzyme. The coenzyme and apoenzyme form holoenzyme. The coenzyme can connect with the protein part either only for the duration of the reaction, or bind to each other with a permanent strong bond (then the non-protein part is called - prosthetic group). In any case, non-protein components are directly involved in chemical reactions by interacting with the substrate. Coenzymes can be represented by:

Nucleoside triphosphates.

Minerals (zinc, copper, magnesium).

Active forms of vitamins (B 1 is part of the enzyme decarboxylase, B 2 is part of dehydrogenase, B 6 is part of transferase).

Main functions of coenzymes:

Participation in the act of catalysis.

Establishing contact between enzyme and substrate.

Stabilization of the apoenzyme.

The apoenzyme, in turn, enhances the catalytic activity of the non-protein part and determines the specificity of the action of enzymes.

Each enzyme contains several functional centers.

Active center- a zone of an enzyme molecule that specifically interacts with the substrate. The active center is represented by functional groups of several amino acid residues; it is here that the attachment and chemical transformation of the substrate occurs.

Allosteric center or regulatory - this is the zone of the enzyme responsible for the addition of activators and inhibitors. This center is involved in the regulation of enzyme activity.

These centers are located in different parts of the enzyme molecule.

It has long been established that all enzymes are proteins and have all the properties of proteins. Therefore, like proteins, enzymes are divided into simple and complex.

Simple enzymes consist only of amino acids - for example, pepsin , trypsin , lysozyme.

Complex enzymes(holoenzymes) have a protein part consisting of amino acids - apoenzyme, and a non-protein part - cofactor. Examples of complex enzymes are succinate dehydrogenase(contains FAD), aminotransferases(contain pyridoxal phosphate), various peroxidases(contain heme), lactate dehydrogenase(contains Zn 2+), amylase(contains Ca2+).

Cofactor, in turn, can be called a coenzyme (NAD+, NADP+, FMN, FAD, biotin) or a prosthetic group (heme, oligosaccharides, metal ions Fe2+, Mg2+, Ca2+, Zn2+).

The division into coenzymes and prosthetic groups is not always clear:
if the connection of the cofactor with the protein is strong, then in this case they speak of the presence prosthetic group,
but if a vitamin derivative acts as a cofactor, then it is called coenzyme, regardless of the strength of the connection.

To carry out catalysis, a complete complex of apoprotein and cofactor is necessary; they cannot carry out catalysis separately. The cofactor is part of the active center and participates in the binding of the substrate or in its transformation.

Like many proteins, enzymes can be monomers, i.e. consist of one subunit, and polymers, consisting of several subunits.

Structural and functional organization of enzymes

The enzyme contains areas that perform different functions:

1. Active center - a combination of amino acid residues (usually 12-16) that provides direct binding to the substrate molecule and carries out catalysis. Amino acid radicals in the active center can be in any combination, with amino acids located nearby that are significantly distant from each other in the linear chain. There are two regions in the active center:

• anchor(contact, binding) – responsible for binding and orientation of the substrate in the active center,
• catalytic– is directly responsible for the implementation of the reaction.

Enzyme structure diagram

Enzymes that contain several monomers may have several active centers according to the number of subunits. Also, two or more subunits can form one active site.

In complex enzymes, functional groups of the cofactor are necessarily located in the active center.

Scheme of formation of a complex enzyme

2. Allosteric center (allos- foreign) is a center for regulating enzyme activity, which is spatially separated from the active center and is not present in all enzymes. Binding to the allosteric center of any molecule (called an activator or inhibitor, as well as an effector, modulator, regulator) causes a change in the configuration of the enzyme protein and, as a consequence, the rate of the enzymatic reaction.

Allosteric enzymes are polymeric proteins; the active and regulatory centers are located in different subunits.

Scheme of the structure of an allosteric enzyme

Such a regulator can be the product of this or one of the subsequent reactions, a reaction substrate or another substance (see “Regulation of enzyme activity”).

Isoenzymes

Isoenzymes are molecular forms of the same enzyme that arise as a result of slight genetic differences in the primary structure of the enzyme, but catalyze the same reaction. Isoenzymes are different affinity to the substrate, maximum speed catalyzed reaction sensitivity to inhibitors and activators, conditions work (optimum pH and temperature).

As a rule, isoenzymes have quaternary structure, i.e. consist of two or more subunits. For example, the dimeric enzyme creatine kinase (CK) is represented by three isoenzyme forms composed of two types of subunits: M (eng. muscle– muscle) and B (eng. brain- brain). Creatine kinase-1 (CK-1) consists of type B subunits and is localized in the brain, creatine kinase-2 (CK-2) - one M- and B-subunit each, active in the myocardium, creatine kinase-3 (CK-3) contains two M subunits, specific for skeletal muscle. Determination of the activity of different CK isoenzymes in blood serum has.

There are also five isoenzymes lactate dehydrogenase(role of LDH) - an enzyme involved in glucose metabolism. The differences between them lie in the different ratio of H subunits. heart- heart) and M (eng. muscle– muscle). Lactate dehydrogenases types 1 (H 4) and 2 (H 3 M 1) are present in tissues with aerobic metabolism (myocardium, brain, renal cortex), have a high affinity for lactic acid (lactate) and convert it into pyruvate. LDH-4 (H 1 M 3) and LDH-5 (M 4) are found in tissues prone to anaerobic metabolism (liver, skeletal muscle, skin, renal medulla), have a low affinity for lactate and catalyze the conversion of pyruvate to lactate. In tissues with intermediate type of metabolism (spleen, pancreas, adrenal glands, lymph nodes) LDH-3 (H 2 M 2) predominates. Determination of the activity of different LDH isoenzymes in blood serum has clinical and diagnostic significance.

Another example of isozymes is the group hexokinase, which attach a phosphate group to hexose monosaccharides and involve them in cellular metabolic reactions. Of the four isoenzymes, hexokinase IV ( glucokinase), which differs from other isoenzymes in its high specificity for glucose, low affinity for it and insensitivity to inhibition by the reaction product.