The mechanism of action of enzymes. Structure and mechanism of action of enzymes Mechanism of action of enzymes theory of intermediates
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Until recently, it was believed that absolutely all enzymes are substances of a protein nature. But in the 1980s catalytic activity was discovered in some low molecular weight RNAs. These enzymes are called ribozymes. The rest, over 2000 currently known enzymes, are of a protein nature and are characterized by all the properties of proteins.
By structure, enzymes are divided into:
1.simple or one-component;
2. complex or two-component (holoenzymes).
Simple enzymes are simple proteins and, when hydrolyzed, break down only into amino acids. Among simple enzymes are hydrolytic enzymes (pepsin, trypsin, urease, etc.).
Complex proteins are complex proteins and, in addition to polypeptide chains, contain a non-protein component ( cofactor). Most enzymes are complex proteins. The protein part of a two-component enzyme is called apoenzyme. Cofactors can have different bond strengths with the apoenzyme. If the cofactor is firmly associated with the polypeptide chain, it is called prosthetic group. There is a covalent bond between the prosthetic group and the apoenzyme.
If the cofactor is easily separated from the apoenzyme and is capable of independent existence, then such a cofactor is called coenzyme.
Between the apoenzyme and the coenzyme, the bonds are weak - hydrogen, electrostatic, etc.
The chemical nature of cofactors is extremely diverse. The role of cofactors in two-component enzymes is played by:
1 - most vitamins (E, K, Q, C, H, B 1, B 2, B 6, B 12, etc.);
2 compounds of nucleotide nature (NAD, NADP, ATP, CoA, FAD, FMN), as well as a number of other compounds;
3 - lipoic acid;
4 - many divalent metals (Mg 2+, Mn 2+, Ca 2+, etc.).
Active site of enzymes.
Enzymes are macromolecular substances, the molecular weight of which reaches several million. Molecules of substrates that interact with enzymes usually have a much smaller size. Therefore, it is natural to assume that not the entire enzyme molecule as a whole interacts with the substrate, but only some part of it, the so-called “active center” of the enzyme.
The active center of an enzyme is a part of its molecule that directly interacts with substrates and participates in the act of catalysis.
The active center of the enzyme is formed at the level of the tertiary structure. Therefore, during denaturation, when the tertiary structure is broken, the enzyme loses its catalytic activity!
The active center, in turn, consists of:
1. catalytic center, which carries out the chemical transformation of the substrate;
2.substrate center ("anchor" or contact area), which ensures the attachment of the substrate to the enzyme, the formation of the enzyme-substrate complex.
It is not always possible to draw a clear line between the catalytic and substrate centers; in some enzymes, they coincide or overlap.
In addition to the active center, in the enzyme molecule there is a so-called. allosteric center. This is a section of the enzyme molecule, as a result of the addition of a certain low molecular weight substance (effector) to which, the tertiary structure of the enzyme changes. This leads to a change in the configuration of the active site and, consequently, to a change in the activity of the enzyme. This is the phenomenon of allosteric regulation of enzyme activity.
Many enzymes are multimers (or oligomers), i.e. consist of two or more protomer subunits (similar to the quaternary structure of a protein).
The bonds between subunits are mostly non-covalent. The enzyme exhibits the maximum catalytic activity precisely in the form of a multimer. Dissociation into protomers sharply reduces the activity of the enzyme.
Enzymes - multimers usually contain a clear number of subunits (2-4), i.e. are di- and tetramers. Although hexa- and octamers (6-8) are known, and trimers and pentamers (3-5) are extremely rare.
Multimeric enzymes can be built from the same or different subunits.
If multimeric enzymes are formed from different types of subunits, they can exist as multiple isomers. Multiple forms of an enzyme are called isoenzymes (isoenzymes or isozymes).
For example, an enzyme consists of 4 subunits of types A and B. It can form 5 isomers: AAAA, AAAB, AABB, ABBB, BBBB. These isomeric enzymes are isoenzymes.
Isoenzymes catalyze the same chemical reaction, usually act on the same substrate, but differ in some physicochemical properties (molecular weight, amino acid composition, electrophoretic mobility, etc.), localization in organs and tissues.
A special group of enzymes are the so-called. multimeric complexes. These are systems of enzymes that catalyze the successive stages of the transformation of a substrate. Such systems are characterized by the strength of the bond and the strict spatial organization of enzymes, which ensures the minimum pathway for the passage of the substrate and the maximum rate of its transformation.
An example is a multienzyme complex that performs the oxidative decarboxylation of pyruvic acid. The complex consists of 3 types of enzymes (M.v. = 4,500,000).
The mechanism of action of enzymes
The mechanism of action of enzymes is as follows. When a substrate is combined with an enzyme, an unstable enzyme-substrate complex is formed. It activates the substrate molecule due to:
1. polarization of chemical bonds in the substrate molecule and redistribution of electron density;
2. deformation of the bonds involved in the reaction;
3. convergence and the necessary mutual orientation of the substrate molecules (S).
The substrate molecule is fixed in the active center of the enzyme in a strained configuration, in a deformed state, which leads to a weakening of the strength of chemical bonds and reduces the level of the energy barrier, i.e. substrate is activated.
In the process of an enzymatic reaction, 4 stages are distinguished:
1 – attachment of a substrate molecule to an enzyme and formation of an enzyme-substrate complex;
2 - change in the substrate under the action of the enzyme, making it available for a chemical reaction, i.e. substrate activation;
3 - chemical reaction;
4 - separation of reaction products from the enzyme.
This can be written as a diagram:
E + SESES* EPE + P
where: Е – enzyme, S – substrate, S* – activated substrate, Р – reaction product.
At the 1st stage, that part of the substrate molecule that does not undergo chemical transformations is attached to the substrate center using weak interactions. For the formation of an enzyme-substrate complex (ES), three conditions must be met, which determine the high specificity of the enzyme action.
Conditions for the formation of an enzyme-substrate complex:
1.structural conformity between the substrate and the active site of the enzyme. According to Fischer, they should fit together, "like a key to a lock." This similarity is provided at the level of the tertiary structure of the enzyme, i.e. spatial arrangement of the functional groups of the active site.
2.electrostatic compliance the active center of the enzyme and the substrate, which is due to the interaction of oppositely charged groups.
3.flexibility of the tertiary structure of the enzyme - "induced conformity". According to the theory of forced or induced fit, a catalytically active configuration of an enzyme molecule can arise only at the moment of attachment of a substrate as a result of its deforming effect according to the “hand-glove” principle.
The mechanism of action of one-component and two-component enzymes is similar.
Both apoenzyme and coenzyme are involved in the formation of the enzyme-substrate complex in complex enzymes. In this case, the substrate center is usually located on the apoenzyme, and the coenzyme takes part directly in the act of chemical transformation of the substrate. At the last stage of the reaction, apoenzyme and coenzyme are released unchanged.
At stages 2 and 3, the transformation of the substrate molecule is associated with the breaking and closing of covalent bonds.
After the implementation of chemical reactions, the enzyme goes into its original state and the reaction products are separated.
The ability of an enzyme to catalyze a specific type of reaction is called specificity.
There are three types of specificity:
1.relative or group specificity- the enzyme acts on a certain type of chemical bond (for example, the enzyme pepsin cleaves a peptide bond);
2.absolute specificity - the enzyme acts only on one strictly defined substrate (for example, the urease enzyme cleaves the amide bond only in urea);
3.stoichiometric specificity- the enzyme acts only on one of the stereoisomers (for example, the glucosidase enzyme ferments only D-glucose, but does not act on L-glucose).
The specificity of the enzyme ensures the orderliness of the course of metabolic reactions.
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Structure, properties and mechanism of action of enzymes
Content
- Structure of enzymes
- The mechanism of action of enzymes
- Enzyme nomenclature
- Enzyme classification
- Enzyme Properties
- Clinical fermentology
- Literature
Brief history of fermentology
The experimental study of enzymes in the 19th century coincided with the study of yeast fermentation processes, which was reflected in the terms "enzymes" and "enzymes". The name enzymes comes from the Latin word fermentatio - fermentation. The term enzymes comes from the concept of en zyme - from yeast. At first, these names were given different meanings, but at present they are synonymous.
The first enzymatic reaction of starch saccharification with malt was studied by the domestic scientist K.S. Kirchhoff in 1814. Subsequently, attempts were made to isolate enzymes from yeast cells (E. Buechner, 1897). At the beginning of the 20th century, L. Michaelis and M. Menten developed the theory of enzymatic catalysis. In 1926, D. Sumner was the first to isolate a purified preparation of the urease enzyme in a crystalline state. In 1966, B. Merrifield succeeded in artificially synthesizing the enzyme RNase.
Structure of enzymes
Enzymes are highly specialized proteins capable of increasing the rate of reaction in living organisms. Enzymes are biological catalysts.
All enzymes are proteins, usually globular. They can refer to both simple and complex proteins. The protein part of the enzyme may consist of one polypeptide chain - monomeric proteins - enzymes (for example, pepsin). A number of enzymes are oligomeric proteins that include several protomers or subunits. Protomers, uniting into an oligomeric structure, are connected spontaneously by weak non-covalent bonds. In the process of association (cooperation), structural changes occur in individual protomers, as a result of which the activity of the enzyme increases markedly. Separation (dissociation) of protomers and their association into an oligomeric protein is a mechanism for regulating enzyme activity.
Subunits (protomers) in oligomers can be either the same or different in primary - tertiary structure (conformation). In the case of combining different protomers into the oligomeric structure of the enzyme, multiple forms of the same enzyme arise - isoenzymes .
Isoenzymes catalyze the same reaction, but differ in the set of subunits, physicochemical properties, electrophoretic mobility, affinity for substrates, activators, and inhibitors. For example, lactate dehydrogenase (LDH) - the enzyme that oxidizes lactic acid to pyruvic acid is a tetramer. It consists of four protomers of two types. One type of protomer is designated H (isolated from cardiac muscle), the second protomer is designated M (isolated from skeletal muscle). There are 5 possible combinations of these protomers in LDH: H 4 , H 3 M, H 2 M 2 , H 1 M 3 , M 4 .
The biological role of isoenzymes.
· Isoenzymes ensure the flow of chemical reactions in accordance with the conditions in different organs. So, the isoenzyme LDH 1 - has a high affinity for oxygen, so it is active in tissues with a high rate of oxidative reactions (erythrocytes, myocardium). The LDH 5 isoenzyme is active in the presence of a high concentration of lactate, most characteristic of the liver tissue
Pronounced organ specificity is used to diagnose diseases of various organs.
Isoenzymes change their activity with age. So, in a fetus with a lack of oxygen, LDH 3 prevails, and with increasing age, an increase in oxygen supply, the proportion of LDH 2 increases.
enzyme activator inhibitor energy
If an enzyme is a complex protein, then it consists of a protein and a non-protein part. The protein part is the high molecular weight thermolabile part of the enzyme and is called apoenzyme . It has a peculiar structure and determines the specificity of enzymes.
The non-protein portion of an enzyme is called cofactor ( coenzyme ). The cofactor is most often metal ions that can bind strongly to the apoenzyme (for example, Zn in the carbonic anhydrase enzyme, Cu in the cytochrome oxidase enzyme). Coenzymes are most often organic substances that are less tightly bound to the apoenzyme. Coenzymes are nucleotides NAD, FAD. coenzyme - low molecular weight, thermostable part of the enzyme. Its role is that it determines the spatial packing (conformation) of the apoenzyme, and determines its activity. Cofactors can transfer electrons, functional groups, participate in the formation of additional bonds between the enzyme and the substrate.
In functional terms, it is customary to distinguish two important sites in the enzyme molecule: the active site and the allosteric site.
Active center - this is a section of the enzyme molecule that interacts with the substrate and participates in the catalytic process. The active center of the enzyme is formed by amino acid radicals that are distant from each other in the primary structure. The active center has a three-dimensional packing, most often it contains
OH groups serine
SH - cysteine
NH 2 lysine
g-COOH glutamic acid
Two zones are distinguished in the active center - the zone of binding with the substrate and the catalytic zone.
Zone binding usually has a rigid structure to which the reaction substrate is complementarily attached. For example, trypsin cleaves proteins in sites rich in the positively charged amino acid lysine, since its binding zone contains negatively charged aspartic acid residues.
catalytic zone - this is a site of the active center that directly acts on the substrate and performs a catalytic function. This zone is more mobile, it is possible to change the relative position of functional groups in it.
In a number of enzymes (often oligomeric), in addition to the active center, there is allosteric plot - a region of the enzyme molecule remote from the active center and interacting not with the substrate, but with additional substances (regulators, effectors). In allosteric enzymes, one subunit may have an active site, while the other has an allosteric site. Allosteric enzymes change their activity in the following way: the effector (activator, inhibitor) acts on the allosteric subunit and changes its structure. Then, a change in the conformation of the allosteric subunit, according to the principle of cooperative changes, indirectly changes the structure of the catalytic subunit, which is accompanied by a change in the activity of the enzyme.
The mechanism of action of enzymes
Enzymes have a number of general catalytic properties:
do not shift the catalytic equilibrium
are not consumed during the reaction
catalyze only thermodynamically real reactions. Such reactions are those in which the initial energy supply of molecules is greater than the final one.
During the reaction, a high energy barrier is overcome. The difference between the energy of this threshold and the initial energy level is the activation energy.
The rate of enzymatic reactions is determined by the activation energy and a number of other factors.
The rate constant of a chemical reaction is determined by the equation:
TO= P* Z* e - ( Ea / RT )
K - reaction rate constant
P - spatial (steric) coefficient
Z is the number of interacting molecules
E a - activation energy
R - gas constant
T - universal absolute temperature
e is the base of natural logarithms
In this equation, Z, e, R, T are constants, and P and Ea are variables. Moreover, there is a direct relationship between the reaction rate and the steric coefficient, and an inverse and power dependence between the rate and activation energy (the lower Ea, the higher the reaction rate).
The mechanism of action of enzymes is reduced to an increase in the steric coefficient by enzymes and a decrease in the activation energy.
Reduction of activation energy by enzymes
For example, the energy of H 2 O 2 splitting without enzymes and catalysts is 18,000 kcal per mol. If platinum is used and high temperature, it drops to 12,000 kcal/mol. With the help of an enzyme catalase the activation energy is only 2,000 kcal/mol.
The decrease in Ea occurs as a result of the formation of intermediate enzyme-substrate complexes according to the scheme: F+ S <=> FS-complex > F + products reactions. For the first time, the possibility of the formation of enzyme-substrate complexes was proved by Michaelis and Menten. Subsequently, many enzyme-substrate complexes have been isolated. To explain the high selectivity of enzymes during interaction with the substrate, it was proposed theory " key And castle" Fisher. According to it, the enzyme interacts with the substrate only when they absolutely correspond to each other (complementarity), like a key and a lock. This theory explained the specificity of enzymes, but did not reveal the mechanisms of their action on the substrate. Later, the theory of induced enzyme-substrate matching was developed - theory Koshland(the "rubber glove" theory). Its essence is as follows: the active center of the enzyme is formed and contains all the functional groups even before interaction with the substrate. However, these functional groups are in an inactive state. At the moment of attachment of the substrate, it induces changes in the position and structure of radicals in the active center of the enzyme. As a result, the active center of the enzyme becomes active under the action of the substrate and, in turn, begins to act on the substrate, i.e. interaction between the active site of the enzyme and the substrate. As a result, the substrate passes into an unstable, unstable state, which leads to a decrease in the activation energy.
The interaction of the enzyme and the substrate may consist in the reactions of nucleophilic substitution, electrophilic substitution, and dehydration of the substrate. A short-term covalent interaction of the functional groups of the enzyme with the substrate is also possible. Basically, there is a geometric reorientation of the functional groups of the active site.
Increasing the steric coefficient by enzymes
The steric coefficient is introduced for reactions involving large molecules with a spatial structure. The steric coefficient shows the proportion of successful collisions of active molecules. For example, it is equal to 0.4 if 4 out of 10 collisions of active molecules led to the formation of a reaction product.
Enzymes increase the steric coefficient, as they change the structure of the substrate molecule into an enzyme-substrate complex, as a result of which the complementarity of the enzyme and substrate increases. In addition, enzymes, due to their active centers, order the arrangement of substrate molecules in space (before interacting with the enzyme, substrate molecules are arranged randomly) and facilitate the reaction.
Enzyme nomenclature
Enzymes have several types of names.
1) Trivial names (trypsin, pepsin)
2) Working nomenclature. In this name of the enzyme there is an ending - aza, which is added:
to the name of the substrate (sucrose, amylase),
to the type of bond on which the enzyme acts (peptidase, glycosidase),
· to the type of reaction, process (synthetase, hydrolase).
3) Each enzyme has a classification name, which reflects the type of reaction, the type of substrate and coenzyme. For example: LDH - L lactate-NAD + - oxidoreductase.
Enzyme classification
The classification of enzymes was developed in 1961. According to the classification, each enzyme is located in a certain class, subclass, subsubclass and has a serial number. In this regard, each enzyme has a digital cipher, in which the first digit indicates the class, the second - the subclass, the third - the subclass, the fourth - the serial number (LDG: 1,1,1,27). All enzymes are classified into 6 classes.
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Liases
5. Isomerases
6. Synthetases (ligases)
Oxidoreductase .
Enzymes that catalyze redox processes. General view of the reaction: A ok + B vos \u003d A vost + B ok. This class of enzymes includes several subclasses:
1 . dehydrogenases, catalyze reactions by removing hydrogen from the oxidized substance. They can be aerobic (transfer hydrogen to oxygen) and anaerobic (transfer hydrogen not to oxygen, but to some other substance).
2. Oxygenases - enzymes that catalyze oxidation by adding oxygen to the oxidized substance. If one oxygen atom is added, monooxygenases are involved, if two oxygen atoms are added, dioxygenases are involved.
3. Peroxidases - enzymes that catalyze the oxidation of substances with the participation of peroxides.
Transferases .
Enzymes that carry out intramolecular and intermolecular transfer of functional groups from one substance to another according to the scheme: AB + C = A + BC. Subclasses of transferases are distinguished depending on the type of transferred groups: aminotransferases, methyltransferases, sulfotransferases, acyltransferases (transfer fatty acid residues), phosphotransferases (transfer phosphoric acid residues).
Hydrolases .
Enzymes of this class catalyze the breaking of a chemical bond with the addition of water at the place of the break, that is, the hydrolysis reaction according to the scheme: AB + HOH = AH + BOH. Subclasses of hydrolases are distinguished depending on the type of broken bonds: peptidases cleave peptide bonds (pepsin), glycosidases - glycosidic bonds (amylase), esterases - ester bonds (lipase).
Liase .
Lyases catalyze the breaking of a chemical bond without the addition of water at the site of the break. In this case, double bonds are formed in the substrates according to the scheme: AB \u003d A + B. Subclasses of lyases depend on which atoms the bond is broken between and which substances are formed. Aldolases break the bond between two carbon atoms (for example, fructose 1,6-di-phosphate aldolase "cuts" fructose and two trioses). Lyases include decarboxylase enzymes (they split off carbon dioxide), dehydratases - “cut out” water molecules.
Isomerases .
Isomerases catalyze the interconversions of different isomers. For example, phosphohexoisimerase converts fructose to glucose. Subclasses of isomerases include mutases (phosphoglucomutase converts glucose-1-phosphate to glucose-6-phosphate), epimerases (for example, convert ribose to xylulose), tautomerases
Synthetases ( ligases ).
Enzymes of this class catalyze the reactions of synthesis of new substances due to the energy of ATP according to the scheme: A + B + ATP \u003d AB. For example, glutamine synthetase combines glutamic acid, NH 3 + with the participation of ATP to form glutamine.
Enzyme Properties
Enzymes, in addition to properties common to inorganic catalysts, have certain differences from inorganic catalysts. These include:
higher activity
higher specificity
milder conditions for catalysis
ability to regulate activity
High catalytic activity enzymes .
Enzymes are characterized by high catalytic activity. For example, one molecule of carbonic anhydrase in one minute catalyzes the formation (or splitting) of 36 million molecules of carbonic acid (H 2 CO 3). The high activity of enzymes is explained by the mechanism of their action: they reduce the activation energy and increase the spatial (steric coefficient). The high activity of enzymes is of great biological importance, consisting in the fact that they provide a high rate of chemical reactions in the body.
High specificity enzymes .
All enzymes have specificity, but the degree of specificity varies from enzyme to enzyme. There are several types of enzyme specificity.
Absolute substrate specificity, in which the enzyme acts on only one specific substance. For example, the enzyme urease only breaks down urea.
Absolute group specificity, in which the enzyme has the same catalytic effect on a group of compounds that are similar in structure. For example, the enzyme alcohol dehydrogenase oxidizes not only C 2 H 5 OH, but also its homologues (methyl, butyl and other alcohols).
Relative group specificity, in which the enzyme catalyzes different classes of organic substances. For example, the enzyme trypsin exhibits peptidase and esterase activity.
Stereochemical specificity (optical specificity), in which only a certain form of isomers is cleaved (D, L forms, b, c, cis - trans isomers). For example, LDH acts only on L-lactate, L-amino acid oxidases act on L-isomers of amino acids.
The high specificity is due to the unique structure of the active site for each enzyme.
thermolability enzymes .
Thermolability - the dependence of enzyme activity on temperature. With an increase in temperature from 0 to 40 degrees, the activity of enzymes increases according to the van't Hoff rule (with an increase in temperature by 10 degrees, the reaction rate increases by 2-4 times). With a further increase in temperature, the activity of enzymes begins to decrease, which is explained by thermal denaturation of the protein molecule of the enzyme. Graphically, the thermodependence of enzymes has the form:
Enzyme inactivation at 0 degrees is reversible, and at high temperatures, inactivation becomes irreversible. This property of enzymes determines the maximum reaction rate under the conditions of human body temperature. The thermolability of enzymes should be taken into account in practical medical activities. For example, when conducting an enzymatic reaction in a test tube, it is necessary to create an optimal temperature. This property of enzymes can be used in cryosurgery, when a complex long-term operation is performed with a decrease in body temperature, which slows down the rate of reactions occurring in the body and reduces oxygen consumption by tissues. It is necessary to store enzymatic preparations at a low temperature. For neutralization, disinfection of microorganisms, high temperatures are used (autoclaving, boiling of instruments).
Photolability .
Photolability - the dependence of enzyme activity on the action of ultraviolet rays. UV radiation causes photodenaturation of protein molecules and reduces the activity of enzymes. This property of enzymes is used in the bactericidal effect of ultraviolet lamps.
Addiction activity from pH.
All enzymes have a certain pH range in which the activity of the enzyme is maximum - the pH optimum. For many enzymes, the optimum is about 7. At the same time, for pepsin, the optimal environment is 1-2, for alkaline phosphatase, about 9. If the pH deviates from the optimum, the activity of the enzyme decreases, as can be seen from the graph. This property of enzymes is explained by a change in the ionization of ionogenic groups in the enzyme molecules, which leads to a change in ionic bonds in the protein molecule of the enzyme. This is accompanied by a change in the conformation of the enzyme molecule, and this, in turn, leads to a change in its activity. Under the conditions of the body, pH - dependence determines the maximum activity of enzymes. This property also finds practical applications. Enzymatic reactions outside the body are carried out at optimum pH. With reduced acidity of gastric juice, a solution of HCl is prescribed for therapeutic purposes.
Addiction speed enzymatic reactions from concentration enzyme And concentration substrate
The dependence of the reaction rate on the concentration of the enzyme and the concentration of the substrate (the kinetics of enzymatic reactions) is shown in the graphs.
schedule 1 schedule 2
In an enzymatic reaction ( F+ S 2 1 FS> 3 F + P) distinguish the speed of three components of the stages:
1 - formation of the enzyme-substrate complex FS,
2 - reverse decay of the enzyme - substrate complex,
3 - decomposition of the enzyme-substrate complex with the formation of reaction products. The rate of each of these reactions obeys the law of mass action:
V 1 \u003d K 1 [F] * [S]
V 2 \u003d K 2 *
V 3 \u003d K 3 *
At the moment of equilibrium, the reaction rate of formation of FS is equal to the sum of its decay rates: V 1 = V 2 + V 3 . Of the three stages of the enzymatic reaction, the third is the most important and slowest, since it is associated with the formation of reaction products. According to the above formula, it is impossible to find the speed V 3, since the enzyme-substrate complex is very unstable, measuring its concentration is difficult. In this regard, Michaelis-Menten introduced Km - the Michaelis constant and transformed the equation for measuring V 3 into a new equation in which there are actually measurable quantities:
V 3 \u003d K 3 * * [S] / Km + [S] or V 3 \u003d V max * [S] / Km + [S]
- the initial concentration of the enzyme
Km is the Michaelis constant.
Physical meaning of Km: TOm = (TO 2 +K 3 ) /TO 1 . It shows the ratio of the rate constants of the decomposition of the enzyme-substrate complex and the rate constant of its formation.
The Michaelis-Menten equation is universal. It illustrates the dependence of the reaction rate on [S]
1. Dependence of the reaction rate on the substrate concentration. This dependence is revealed at low concentrations of the substrate [S]
V 3
=
K 3*
[
F 0
]
*
[
S]
/
km.
In this equation K 3
,
F 0
],
km -
constants and can be replaced by a new constant K*. Thus, at a low substrate concentration, the reaction rate is directly proportional to this concentration.
V 3
=
K*
*
[
S].
This dependence corresponds to the first section of graph 2.
2. The dependence of the rate on the concentration of the enzyme manifests itself at a high concentration of the substrate.
S?Km.
In this case, Km can be neglected and the equation becomes:
V 3
=
K 3*
(([
F 0
]
*
[
S])
/
[
S])
=
K 3*
[
F 0
]
=
V max.
Thus, at a high substrate concentration, the reaction rate is determined by the enzyme concentration and reaches its maximum value
V 3
=
K 3
[
F 0
]
=
V max.
( third section of graph 2).
3. Allows you to determine the numerical value of Km under the condition V 3 = V max /2. In this case, the equation takes the form:
V max /2 = ((V max * [S]) / Km+ [S]), whence it follows that Km= [S]
Thus, Km is numerically equal to the concentration of the substrate at a reaction rate equal to half the maximum. Km is a very important characteristic of an enzyme, it is measured in moles (10 -2 - 10 -6 mol) and characterizes the specificity of the enzyme: the lower the Km, the higher the specificity of the enzyme.
Graphic
definition
constants
Michaelis.
It is more convenient to use a graph representing a straight line.
Such a graph is proposed by Lineweaver - Burke (plot of double reciprocals), which corresponds to the inverse Michaelis - Menten equation
Dependence of the rate of enzymatic reactions on the presence of activators and inhibitors
Activators
- substances that increase the rate of enzymatic reactions. There are specific activators that increase the activity of one enzyme (HCl - pepsinogen activator) and non-specific activators that increase the activity of a number of enzymes (Mg ions - activators of hexokinase, K, Na - ATPase and other enzymes). Metal ions, metabolites, nucleotides can serve as activators.
The mechanism of action of activators
1. Completion of the active center of the enzyme, as a result of which the interaction of the enzyme with the substrate is facilitated. This mechanism is mainly possessed by metal ions.
2. The allosteric activator interacts with the allosteric site (subunit) of the enzyme, through its changes indirectly changes the structure of the active center and increases the activity of the enzyme. The metabolites of enzymatic reactions, ATP, have an allosteric effect.
3. The allosteric mechanism can be combined with a change in the oligomerism of the enzyme. Under the action of the activator, several subunits are combined into an oligomeric form, which dramatically increases the activity of the enzyme. For example, isocitrate is an activator of the enzyme acetyl-CoA carboxylase.
4. Phospholylation - dephosphorylation of enzymes refers to the reversible modification of enzymes. The addition of H 3 RO 4 most often sharply increases the activity of the enzyme. For example, two inactive dimers of the phosphorylase enzyme combine with four ATP molecules to form the active tetrameric phosphorylated form of the enzyme. The phosphorylation of enzymes can be combined with a change in their oligomericity. In some cases, phosphorylation of the enzyme, on the contrary, reduces its activity (for example, phosphorylation of the enzyme glycogen synthetase)
5. Partial proteolysis (irreversible modification). With this mechanism, a fragment of the molecule is cleaved off from the inactive form of the enzyme (proenzyme), blocking the active center of the enzyme. For example, inactive pepsinogen is converted to active pepsin by HCL.
Inhibitors
-
substances that reduce enzyme activity.
By
specificity distinguish between specific and non-specific inhibitors
By
reversibility effect distinguish between reversible and irreversible inhibitors.
By
place
actions there are inhibitors that act on the active center and outside the active center.
By
mechanism
actions distinguish between competitive and non-competitive inhibitors.
Competitive
inhibition
.
Inhibitors of this type have a structure close to that of the substrate. Because of this, inhibitors and the substrate compete for the binding of the active site of the enzyme. Competitive inhibition is reversible inhibition The effect of a competitive inhibitor can be reduced by increasing the concentration of the reaction substrate
An example of competitive inhibition is the inhibition of the activity of succinate dehydrogenase, which catalyzes the oxidation of dicarboxylic succinic acid, by dicarboxylic malonic acid, similar in structure to succinic acid.
The principle of competitive inhibition is widely used in the development of drugs. For example, sulfanilamide preparations have a structure close to the structure of para-aminobenzoic acid, which is necessary for the growth of microorganisms. Sulfonamides block the enzymes of microorganisms necessary for the absorption of para-aminobenzoic acid. Some anticancer drugs are analogues of nitrogenous bases and thus inhibit the synthesis of nucleic acids (fluorouracil).
Graphically, competitive inhibition has the form:
Non-competitive
inhibition
.
Non-competitive inhibitors are not structurally similar to reaction substrates and therefore cannot be displaced at high substrate concentrations. There are several options for the action of non-competitive inhibitors:
1. Blocking of the functional group of the active center of the enzyme and, as a result, a decrease in activity. For example, the activity of SH - groups can bind thiol poisons reversibly (salts of metals, mercury, lead) and irreversibly (monioiodoacetate). The inhibition effect of thiol inhibitors can be reduced by the introduction of additional substances rich in SH groups (for example, unithiol). There are and are used serine inhibitors that block the OH - groups of the active center of enzymes. Organic phosphofluorine-containing substances have this effect. These substances can, in particular, inhibit OH groups in the enzyme acetylcholinesterase, which destroys the neurotransmitter acetylcholine.
2. Blocking of metal ions that are part of the active center of enzymes. For example, cyanides block iron atoms, EDTA (ethylenediaminetetraacetate) blocks Ca, Mg ions.
3. An allosteric inhibitor interacts with the allosteric site, indirectly through it according to the principle of cooperativity, changing the structure and activity of the catalytic site. Graphically, non-competitive inhibition has the form:
The maximum reaction rate in non-competitive inhibition cannot be achieved by increasing the substrate concentration.
Regulation of enzyme activity during metabolism
Adaptation of the body to changing conditions (diet, environmental impacts, etc.) is possible due to a change in the activity of enzymes. There are several possibilities for regulating the rate of enzymatic reactions in the body:
1. Change in the rate of enzyme synthesis (this mechanism requires a long period of time).
2. Increasing the availability of the substrate and the enzyme by changing the permeability of cell membranes.
3. Change in the activity of enzymes already present in cells and tissues. This mechanism is carried out at high speed and is reversible.
In multistage enzymatic processes, regulatory, key enzymes are isolated that limit the overall rate of the process. Most often, these are enzymes of the initial and final stages of the process. Changes in the activity of key enzymes occur through various mechanisms.
1. Allosteric mechanism:
2. Change in enzyme oligomerism:
Monomers are inactive - oligomers are active
3. Phospholylation - dephosphorylation:
Enzyme (inactive) + H 3 RO 4 - phosphorylated active enzyme.
Autoregulatory mechanism is widespread in cells. The autoregulatory mechanism is, in particular, retroinhibition, in which the products of the enzymatic process inhibit the enzymes of the initial stages. For example, high concentrations of purine and pyrimidine nucleotides inhibit the initial ones at the stage of their synthesis.
Sometimes the initial substrates activate the final enzymes, in the scheme: substrate A activates F 3 . For example, the active form of glucose (glucose-6-phosphate) activates the final enzyme in the synthesis of glycogen from glucose (glycogen synthetase).
Structural organization of enzymes in a cell
The coherence of metabolic processes in the body is possible due to the structural dissociation of enzymes in cells. Individual enzymes are located in certain intracellular structures - compartmentalization
.
For example, the enzyme potassium, sodium ATPase, is active in the plasma membrane. In mitochondria, enzymes of oxidative reactions (succinate dehydrogenase, cytochrome oxidase) are active. Enzymes for the synthesis of nucleic acids (DNA polymerase) are active in the nucleus. In lysosomes, enzymes for the breakdown of various substances (RNase, phosphatase, and others) are active.
Enzymes that are most active in a given cell structure are called indicator
or marker enzymes. Their definition in clinical practice reflects the depth of structural tissue damage. Some enzymes combine into polyenzymatic complexes, for example, the pyruvate dehydrogenase complex (PDC), which oxidizes pyruvic acid.
PrinciplesdetectionAndquantitativedefinitionsenzymes:
The detection of enzymes is based on their high specificity. Enzymes are detected by the action they produce, i.e. upon the occurrence of the reaction catalyzed by the enzyme. For example, amylase is detected by the breakdown of starch to glucose.
The criteria for the occurrence of an enzymatic reaction can be:
disappearance of the reaction substrate
appearance of reaction products
change in the optical properties of the coenzyme.
Quantification of enzymes
Since the concentration of enzymes in cells is very low, it is not their true concentration that is determined, but the amount of the enzyme is judged indirectly, by the activity of the enzyme.
Enzyme activity is assessed by the rate of the enzymatic reaction occurring under optimal conditions (optimal temperature, pH, excessively high concentration of the substrate). Under these conditions, the reaction rate is directly proportional to the concentration of the enzyme (V= K 3 ).
Units
activity
(
quantity
)
enzyme
In clinical practice, several units of enzyme activity are used.
1. International unit - the amount of enzyme that catalyzes the conversion of 1 micromol of substrate per minute at a temperature of 25 0 C.
2. Catal (in the SI system) - the amount of enzyme that catalyzes the transformation of 1 mole of the substrate per second.
3. Specific activity - the ratio of enzyme activity to the mass of enzyme protein.
4. The molecular activity of an enzyme shows how many substrate molecules are converted under the action of 1 enzyme molecule.
Clinical fermentology
The use of information about enzymes in medical practice is a branch of medical enzymology. It includes 3 sections:
1. Enzymodiagnostics
2. Enzymopotology
3. Enzyme therapy
Enzymodiagnostics
-
a section that studies the possibilities of studying the activity of enzymes for diagnosing diseases. To assess the damage to individual tissues, organ-specific enzymes, isoenzymes are used.
In pediatric practice, when conducting enzyme diagnostics, it is necessary to take into account children's characteristics. In children, the activity of some enzymes is higher than in adults. For example, high LDH activity reflects the predominance of anaerobic processes in the early postnatal period. The content of transaminases in the blood plasma of children is increased as a result of increased vascular tissue permeability. The activity of glucose-6-phosphate dehydrogenase is increased as a result of increased breakdown of erythrocytes. The activity of other enzymes, on the contrary, is lower than in adults. For example, the activity of pepsin, pancreatic enzymes (lipase, amylase) is reduced due to the immaturity of secretory cells.
With age, redistribution of individual isoenzymes is possible. So, in children LDH 3 (more anaerobic form) prevails, and in adults LDH 2 (more aerobic form).
Enzymopathology
- a branch of fermentology that studies diseases, the leading mechanism of development of which is a violation of the activity of enzymes. These include metabolic disorders of carbohydrates (galactosemia, glycogenosis, mucopolysaccharidoses), amino acids (phenylketonuria, cystinuria), nucleotides (orotataciduria), porphyrins (porphyrias).
enzyme therapy
-
branch of fermentology that studies the use of enzymes, coenzymes, activators, inhibitors for therapeutic purposes. Enzymes can be used with a substitution purpose (pepsin, pancreatic enzymes), with a lytic purpose to remove necrotic masses, blood clots, to thin viscous exudates.
Literature
1. Avdeeva, L.V. Biochemistry: Textbook / L.V. Avdeeva, T.L. Aleinikova, L.E. Andrianova; Under the editorship of E.S. Severin. - M.: GEOTAR-MED, 2013. - 768 p.
2. Auerman, T.L. Fundamentals of Biochemistry: Textbook / T.L. Auerman, T.G. Generalova, G.M. Suslyanok. - M.: NITs INFRA-M, 2013. - 400 p.
3. Bazarnova Yu.G. Biochemical bases of processing and storage of raw materials of animal origin: Textbook / Yu.G. Bazarnova, T.E. Burova, V.I. Marchenko. - St. Petersburg: Ave. Science, 2011. - 192 p.
4. Baishev, I.M. Biochemistry. Test questions: Textbook / D.M. Zubairov, I.M. Baishev, R.F. Baykeev; Under the editorship of D.M. Zubairov. - M.: GEOTAR-Media, 2008. - 960 p.
5. Bokut, S.B. Biochemistry of phylogenesis and ontogenesis: Textbook / A.A. Chirkin, E.O. Danchenko, S.B. Bokut; Under total ed.A. A. Chirkin. - M.: NITs INFRA-M, Nov. knowledge, 2012. - 288 p.
6. Gidranovich, V.I. Biochemistry: Textbook / V.I. Gidranovich, A.V. Gidranovich. - Minsk: TetraSystems, 2012. - 528 p.
7. Goloshchapov, A.P. Genetic and biochemical aspects of human adaptation to the conditions of a city with a developed chemical industry / A.P. Goloshchapov. - M.: KMK, 2012. - 103 p.
8. Gunkova, P.I. Biochemistry of milk and dairy products / K.K. Gorbatova, P.I. Gunkov; Under total ed.K. K. Gorbatov. - St. Petersburg: GIORD, 2010. - 336 p.
9. Dimitriev, A.D. Biochemistry: Textbook / A.D. Dimitriev, E.D. Ambrosiev. - M.: Dashkov i K, 2013. - 168 p.
10. Ershov, Yu.A. General biochemistry and sport: Textbook / Yu.A. Ershov. - M.: MGU, 2010. - 368 p.
11. Ershov, Yu.A. Fundamentals of Biochemistry for Engineers: Textbook / Yu.A. Ershov, N.I. Zaitsev; Under the editorship of S.I. Schukin. - M.: MSTU im. Bauman, 2010. - 359 p.
12. Kamyshnikov, V.S. Handbook of clinical and biochemical laboratory diagnostics: In 2 volumes. In 2 volumes. Handbook of clinical and biochemical laboratory diagnostics: In 2 volumes / V.S. Kamyshnikov. - Minsk: Belarus, 2012. - 958 p.
13. Klopov, M.I. Biologically active substances in physiological and biochemical processes in the animal body: Textbook / M.I. Klopov, V.I. Maksimov. - St. Petersburg: Lan, 2012. - 448 p.
14. Mikhailov, S.S. Sports biochemistry: A textbook for universities and colleges of physical culture / S.S. Mikhailov. - M.: Sov. sport, 2012. - 348 p.
15. Repnikov, B.T. Commodity science and biochemistry of fish products: Textbook / B.T. Repnikov. - M.: Dashkov i K, 2013. - 220 p.
16. Rogozhin, V.V. Biochemistry of milk and meat: Textbook / V.V. Rogozhin. - St. Petersburg: GIORD, 2012. - 456 p.
17. Rogozhin, V.V. Plant Biochemistry: Textbook / V.V. Rogozhin. - St. Petersburg: GIORD, 2012. - 432 p.
18. Rogozhin, V.V. Workshop on plant physiology and biochemistry: Textbook / V.V. Rogozhin, T.V. Rogozhin. - St. Petersburg: GIORD, 2013. - 352 p.
19. Taganovich, A.D. Pathological biochemistry: Monograph / A.D. Taganovich. - M.: BINOM, 2013. - 448 p.
20. Filippovich, Yu.B. Biochemical foundations of human life: Textbook for university students / Yu.B. Filippovich, A.S. Konichev, G.A. Sevastyanova, N.M. Kutuzov. - M.: VLADOS, 2005. - 407 p.
21. Shcherbakov, V.G. Biochemistry and commodity science of oil raw materials / V.G. Shcherbakov, V.G. Lobanov. - M.: KolosS, 2012. - 392 p.
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ChapterIV.3.
Enzymes
Metabolism in the body can be defined as the totality of all chemical transformations undergone by compounds coming from outside. 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 proceed in the body at an extremely high rate only in the presence of catalysts. All biological catalysts are substances of a 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 transformations. The 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 wide variety of reactions in the body. So, quite simple from the point of view of traditional chemistry, the reaction of splitting off 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 the pH of the blood. Thanks to the catalytic action of enzymes in the body, it becomes possible to carry out such reactions that would go hundreds and thousands of times slower without a catalyst.
Enzyme Properties
1. Influence on the rate of a chemical reaction: enzymes increase the rate of a chemical reaction, but they themselves are not consumed.
The reaction rate is the change in the concentration of the reaction components per unit time.
If it goes in the forward direction, then it is proportional to the concentration of the reactants; if it goes 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 in the presence of enzymes comes faster.
2. The specificity of the action of enzymes. In the cells of the body, 2-3 thousand reactions take place, each of which is catalyzed by a certain enzyme. The specificity of the action of an enzyme is the ability to accelerate the course of one particular reaction without affecting the rate of others, even very similar ones.
Distinguish:
Absolute– when F catalyzes only one specific reaction ( arginase- breakdown of arginine)
Relative(group special) - F catalyzes a certain class of reactions (eg 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. The activity of enzymes is the ability to accelerate the reaction rate to varying degrees. Activity is expressed in:
1) International units of activity - (IU) the amount of the enzyme catalyzing the conversion of 1 μM of the substrate in 1 min.
2) Katalakh (cat) - the amount of catalyst (enzyme) capable of converting 1 mol of substrate in 1 s.
3) Specific activity - the number of units of activity (any of the above) in the test sample to the total mass of protein in this sample.
4) Less often, molar activity is used - the number of substrate molecules converted by one enzyme molecule per minute.
activity depends on temperature
. This or that enzyme shows the greatest activity at an optimum temperature. For F of a living organism, this value is within +37.0 - +39.0°
C, depending on the type of animal. With a decrease in temperature, 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. In case of temperature increase above +40 - +50°
With the enzyme molecule, which is a protein, undergoes a process of denaturation. At the same time, the rate of the chemical reaction drops noticeably (Fig. 4.3.1.).
Enzyme activity also depends on medium pH
. For most of them, there is a certain optimal pH value at which their activity is maximum. Since the cell contains hundreds of enzymes and each of them has its own opt pH limits, the change in pH is 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 opt of which lies in the range of 7.0 - 7.2, a product is formed, which is an acid. In this case, the pH value shifts to the region of 5.5 - 6.0. The activity of the enzyme sharply decreases, the rate of product formation slows down, but 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).
The chemical nature of enzymes. The structure of the enzyme. Active and allosteric centers
All enzymes are proteins with a molecular weight of 15,000 to several million Da. According to the chemical structure, they are simple enzymes (consist only of AA) and complex enzymes (have a non-protein part or a prosthetic group). The protein portion is called apoenzyme,
and non-protein, if it is covalently linked to an apoenzyme, then 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 a 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 catalysis of the 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 when the protein molecule is twisted into a tertiary structure. Separate sections of amino acids approach each other, forming a certain configuration of the active center. An important structural feature of the active center is that its surface is complementary to the surface of the substrate; AA residues of this zone of the enzyme are able to enter into chemical interaction with certain groups of the substrate. It can be imagined that the active site of the enzyme matches the structure of the substrate like a key and a lock.
IN active center two zones are distinguished: binding center, responsible for the attachment of the substrate, and catalytic center responsible for the chemical transformation of the substrate. The composition of the catalytic center of most enzymes includes such AAs as Ser, Cys, His, Tyr, Lys. Complex enzymes in the catalytic center have a cofactor or coenzyme.
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.
The mechanism of action of enzymes
The act of catalysis consists of three successive stages.
1.
Formation of an enzyme-substrate complex during interaction through the active center.
2.
The binding of the substrate occurs at several points of the active center, which leads to a change in the structure of the substrate, its deformation due to a change in the bond energy in the molecule. This is the second stage and is called substrate activation. When this occurs, a certain chemical modification of the substrate and its transformation into a new product or products.
3.
As a result of such a transformation, the new substance (product) loses its ability to be retained in the active center of the enzyme and the enzyme-substrate, or rather, the enzyme-product complex, dissociates (disintegrates).
Types of catalytic reactions:
A + E \u003d AE \u003d BE \u003d E + B
A + B + E \u003d AE + B \u003d ABE \u003d AB + E
AB + E \u003d ABE \u003d A + B + E, where E is an 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 are distinguished inhibitors
- slowing down the rate of reaction and activators
- accelerating the enzymatic reaction.
Depending on the mechanism of inhibition of the reaction, 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 with a lock (or almost coincides). The degree of this similarity may even be higher than with the substrate.
If A + E \u003d AE \u003d BE \u003d E + B, then I + E \u003d IE¹
The concentration of the enzyme capable of catalysis decreases and the rate of formation of reaction products drops sharply (Fig. 4.3.2.).
![](https://i1.wp.com/test.kirensky.ru/books/book/biochemistry/chapter_03.files/image007.gif)
A large number of chemicals of endogenous and exogenous origin (i.e., 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, maybe. they inhibit key metabolic reactions of microorganisms (sulfonamides) and tumor cells. But with an excess of the substrate and a low concentration of a competitive inhibitor, its action is canceled.
The second type of inhibitors is non-competitive. They interact with the enzyme outside the active site, and an excess of 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.
There are also reversible and irreversible inhibition. Irreversible inhibitors inactivate the enzyme by forming a chemical bond with its AA or other structural components. Usually this is a covalent bond with one of the sites of the active center. Such a complex practically does not dissociate under physiological conditions. In another case, the inhibitor disrupts the conformational structure of the enzyme molecule - causing its denaturation.
The action of reversible inhibitors can be removed by an excess of the substrate or by the action 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 effects,
2)
form a complex with the substrate, which more actively binds to the active center of F,
3)
interacting with an enzyme having a quaternary structure, they separate its subunits and thereby open 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. So 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 peculiar only to 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 the 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 related stages of metabolism occur.
Many enzymes are formed in cells and secreted into the anatomical cavities in an inactive state - these are proenzymes. Often in the form of proenzymes, proteolytic enzymes (break down proteins) are formed. Then, under the influence of pH or other enzymes and substrates, their chemical modification occurs and the active center becomes available 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.
the name of the substrate with which it interacts
2.
the nature of the catalyzed reaction
3.
the 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 must be classified. Currently, an international classification of enzymes has been adopted, which is based on the type of catalyzed reaction. There are 6 classes, which in turn are divided into a number of subclasses (in this book they are presented only selectively):
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 B 2, B 5. 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 cleaved off by the enzyme, turning into the reduced form (NADH, NADPH, FADH) and transfer it to another enzyme-substrate complex, where it is given away.
1.2.
Oxidase - catalyzes the transfer of hydrogen to oxygen with the formation of water or H 2 O 2. F. Cytochromoxysdase respiratory chain.
RH + NAD H + O 2 = ROH + NAD + H 2 O
1.3.
Monooxidases - cytochrome P450. According to its structure, both hemo- and flavoprotein. It hydroxylates lipophilic xenobiotics (by the mechanism described above).
1.4.
PeroxidasesAnd catalase- catalyze the decomposition of hydrogen peroxide, which is formed during metabolic reactions.
1.5.
Oxygenases - catalyze the reactions of oxygen addition to the substrate.
2.
Transferases
- catalyze the transfer of various radicals from the donor molecule to the 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 (acyl group transfer - R-C=O).
Example: synthesis of the neurotransmitter acetylcholine (see chapter "Protein metabolism").
2.3.
Hexosyl transferases catalyze the transfer of glycosyl residues.
Example: the splitting of a glucose molecule from glycogen under the action of phosphorylase.
2.4.
Aminotransferases - transfer of amino groups
R 1- CO - R 2 + R 1 - CH - NH 3
- R 2 \u003d R 1 - CH - NH 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.
Phosphotransferesis (kinase) - catalyze the transfer of a phosphoric acid residue. In most cases, ATP is the phosphate donor. Enzymes of this class are mainly involved in the process of glucose breakdown.
Example: Hexo (gluco) kinase.
3.
Hydrolases
- catalyze hydrolysis reactions, i.e. splitting of substances with addition at the place of breaking the bond of water. This class includes mainly digestive enzymes, they are one-component (do not contain a non-protein part)
R1-R2 + H 2 O \u003d R1H + R2OH
3.1.
Esterases - break down essential bonds. This is a large subclass of enzymes that catalyze the hydrolysis of thiol esters, phosphoesters.
Example: NH 2 ).
Example: arginase(urea cycle).
4. Liases
- catalyze the reactions of cleavage of molecules without the addition of 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 commonly referred to as 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 the synthesis of more complex substances from simple ones. Such reactions proceed with the expenditure of ATP energy. Synthetase is added to the name of such enzymes.
LITERATURE TO THE CHAPTER IV.3.
1. Byshevsky A. Sh., Tersenov O. A. Biochemistry for a doctor // Ekaterinburg: Ural worker, 1994, 384 p.;
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.: Prosveschenie, 1982, 311 pp.;
4. Lehninger A. Biochemistry. Molecular bases of the structure and functions of the cell // M.: Mir, 1974, 956 p.;
5. Pustovalova L.M. Workshop on biochemistry // Rostov-on-Don: Phoenix, 1999, 540 p.
The mechanism of action of enzymes
The mechanism of action of enzymes can be considered from two positions: from the point of view of changes in the energy of chemical reactions and from the point of view of events in the active center.
A. Energy changes in chemical reactions
Any chemical reactions proceed, obeying two basic laws of thermodynamics: the law of conservation of energy and the law of entropy. According to these laws, the total energy of a chemical system and its environment remains constant, while the chemical system tends to reduce order (increase entropy). To understand the energy of a chemical reaction, it is not enough to know the energy balance of the reactants entering and exiting the reaction; it is necessary to take into account the energy changes in the course of a given chemical reaction and the role of enzymes in the dynamics of this process. Consider the decomposition reaction of carbonic acid:
H 2 CO 3 > H 2 0 + C0 2.
Carbonic acid is weak; the reaction of its decomposition will proceed under normal conditions, if the molecules of carbonic acid have an energy exceeding a certain level, called the activation energy E a (Fig. 2-10).
The activation energy is the additional amount of kinetic energy required by the molecules of a substance for them to react.
When this energy barrier is reached, changes occur in the molecule that cause the redistribution of chemical bonds and the formation of new compounds. Molecules with E a are said to be in a transition state. The energy difference between the initial reagent H 2 CO 3 and the final compounds H 2 O and CO 2 is called the change in free
![](https://i1.wp.com/studwood.ru/imag_/43/155548/image002.jpg)
Rice. 2-10. Change in free energy during the decomposition of carbonic acid.
Reaction energies DG. H 2 O and CO 2 molecules are more stable substances than H 2 CO 3 , i.e. have less energy and practically do not react under normal conditions. The released energy as a result of this reaction is dissipated in the form of heat into the environment.
The more molecules have an energy exceeding the level of E a, the higher the rate of a chemical reaction. The rate of a chemical reaction can be increased by heating. This increases the energy of the reacting molecules. However, high temperatures are detrimental to living organisms, so enzymes are used in the cell to speed up chemical reactions. Enzymes provide a high rate of reactions under optimal conditions existing in the cell, by lowering the level of Ea. Thus, enzymes lower the height of the energy barrier, as a result, the number of reactive molecules increases, therefore, the reaction rate increases.
In the mechanism of enzymatic catalysis, the formation of unstable intermediates is of decisive importance - the enzyme-substrate complex ES, which undergoes transformation into an unstable transition complex EP, which almost instantly decomposes into a free enzyme and a reaction product.
Thus, biological catalysts (enzymes) do not change the free energy
substrates and products and therefore do not change the equilibrium of the reaction (Fig. 2-11).
The enzyme, acting as a catalyst for a chemical reaction, obeys the general laws of catalysis and has all the properties characteristic of non-biological catalysts, however, it also has distinctive properties associated with the structural features of enzymes.
Enzymes are similar to non-biological catalysts in that:
- Enzymes catalyze energetically possible reactions;
- the energy of a chemical system remains constant;
- during catalysis, the direction of the reaction does not change;
- Enzymes are not consumed during the reaction.
The differences between enzymes and non-biological catalysts are that:
- · the rate of enzymatic reactions is higher than the reactions catalyzed by non-protein catalysts;
- Enzymes have high specificity;
- The enzymatic reaction takes place in the cell, i.e. at a temperature of 37 °C, constant atmospheric pressure and physiological pH value;
- The rate of the enzymatic reaction can be controlled.
1. Formation of the enzyme-substrate complex
The fact that enzymes have high specificity made it possible in 1890 to put forward a hypothesis according to which the active center of the enzyme is complementary to the substrate, i.e. corresponds to it as "the key to the lock". After the interaction of the substrate ("key") with the active center ("lock"), chemical transformations of the substrate into the product occur. The active center was considered as a stable, rigidly determined structure.
In 1959, another variant of the "key-lock" hypothesis was proposed to explain the events in the active site of the enzyme. According to this hypothesis, the active center is a flexible structure
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Rice. 2-11. The change in free energy during a chemical reaction, uncatalyzed and catalyzed by enzymes.
The enzyme lowers the activation energy E a, i.e. reduces the height of the energy barrier, as a result, the proportion of reactive molecules increases, therefore, the reaction rate increases with respect to the substrate. The substrate, interacting with the active site of the enzyme, causes a change in its conformation, leading to the formation of an enzyme-substrate complex that is favorable for chemical modifications of the substrate. In this case, the substrate molecule also changes its conformation, which ensures a higher efficiency of the enzymatic reaction. This "hypothesis of induced fit" subsequently received experimental confirmation.
2. Sequence of events during enzymatic catalysis
The process of enzymatic catalysis can be conditionally divided into the following stages (Fig. 2-12). substrate catalysis chemical reaction
The first, second, and fourth stages of catalysis are short and depend on the substrate concentration (for the first stage) and ligand binding constants in the active site of the enzyme (for the first and third stages). Changes in the energy of the chemical reaction at these stages are insignificant.
The third stage is the slowest; its duration depends on the activation energy of the chemical reaction. At this stage, bonds are broken in the substrate molecule, new bonds are formed, and the product molecule is formed.
3. Role of the active site in enzymatic catalysis
As a result of research, it was shown that the enzyme molecule, as a rule, is many times larger than the substrate molecule undergoing chemical transformation by this enzyme. Only a small part of the enzyme molecule comes into contact with the substrate, usually from 5 to 10 amino acid residues, which form the active site of the enzyme. The role of the remaining amino acid residues is to ensure the correct conformation of the enzyme molecule for the optimal course of the chemical reaction.
The active site at all stages of enzymatic catalysis cannot be considered as a passive site for substrate binding. It is a complex molecular "machine" that uses a variety of chemical mechanisms to promote the transformation of a substrate into a product.
In the active center of the enzyme, the substrates are arranged in such a way that the functional groups of the substrates participating in the reaction are in close proximity to each other. This property of the active center is called the effect of approach and orientation of the reactants. Such an ordered arrangement of substrates causes a decrease in entropy and, as a consequence, a decrease in the activation energy (E a), which determines the catalytic efficiency of enzymes.
The active center of the enzyme also contributes to the destabilization of interatomic bonds in the substrate molecule, which facilitates the course of a chemical reaction and the formation of products. This property of the active site is called the substrate deformation effect (Fig. 2-12).
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Rice. 2-12. Stages of enzymatic catalysis.
I - stage of approach and orientation of the substrate relative to the active center of the enzyme; II - the formation of an enzyme-substrate complex (ES) as a result of induced compliance; III - deformation of the substrate and the formation of an unstable enzyme-product complex (EP); IV- disintegration of the complex (EP) with the release of reaction products from the active center of the enzyme and the release of the enzyme.
B. Molecular mechanisms of enzymatic catalysis
The mechanisms of enzymatic catalysis are determined by the role of the functional groups of the active center of the enzyme in the chemical reaction of the transformation of the substrate into the product. There are 2 main mechanisms of enzymatic catalysis: acid-base catalysis and covalent catalysis.
1. Acid-base catalysis
The concept of acid-base catalysis explains enzymatic activity by the participation of acid groups (proton donors) and/or basic groups (proton acceptors) in a chemical reaction. Acid-base catalysis is a common phenomenon. The amino acid residues that make up the active center have functional groups that exhibit the properties of both acids and bases.
The amino acids involved in acid-base catalysis primarily include Cys, Tyr, Ser, Lys, Glu, Asp, and His. The radicals of these amino acids in the protonated form are acids (proton donors), in the deprotonated form they are bases (proton acceptors). Due to this property of functional groups of the active site, enzymes become unique biological catalysts, in contrast to non-biological catalysts that can exhibit either acidic or basic properties.
An example of acid-base catalysis, in which Zn 2+ ions are cofactors, and the NAD + molecule is used as a coenzyme, is the liver alcohol dehydrogenase enzyme, which catalyzes the alcohol oxidation reaction (Fig. 2-13):
C 2 H 5 OH + NAD + > CH 3 -SON + NADH + H
2. Covalent catalysis
Covalent catalysis is based on the attack of nucleophilic (negatively charged) or electrophilic (positively charged) groups of the active center of the enzyme by substrate molecules with the formation of a covalent bond between the substrate and the coenzyme or the functional group of the amino acid residue (usually one) of the active center of the enzyme.
The action of serine proteases such as trypsin, chymotrypsin and thrombin is an example of the mechanism of covalent catalysis, when a covalent bond is formed between the substrate and the serine amino acid residue of the active site of the enzyme. The term "serine proteases" is due to the fact that the amino acid residue of serine is part of the active center of all these enzymes and is directly involved in catalysis. Let us consider the mechanism of covalent catalysis using the example of chymotrypsin, which hydrolyzes peptide bonds during protein digestion in the duodenum (see Section 9). The substrates of chymotrypsin are peptides containing amino acids with aromatic and cyclic
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Rice. 2-13. The mechanism of acid-base catalysis on the example of liver alcohol dehydrogenase.
I - an ethanol molecule has a binding center that provides hydrophobic interaction between the active center and the methyl group of alcohol; II - a positively charged zinc atom contributes to the elimination of a proton from the alcohol group of ethanol with the formation of a negatively charged oxygen atom. The negative charge is redistributed between the oxygen atom and the neighboring hydrogen atom, which is then transferred in the form of hydrithione to the fourth carbon atom of the NAD+ coenzyme nicotinamide; III - as a result, the reduced form of NADH and acetaldehyde are formed.
Hydrophobic radicals (Phen, Tyr, Tri), which indicates the participation of hydrophobic forces in the formation of the enzyme-substrate complex. The mechanism of covalent catalysis of chymotrypsin is considered in fig. 2-14.
The radicals Asp 102, Gis 57, and Ser 195 are directly involved in the act of catalysis. Due to the nucleophilic attack of the peptide bond of the substrate, this bond is broken with the formation of a covalently modified serine - acyl-chymotrypsin. Another peptide fragment is released as a result of breaking the hydrogen bond between the peptide fragment and His 57 of the chymotrypsin active site. The final step in the hydrolysis of the peptide bond of proteins is the deacylation of chymotrypsin in the presence of a water molecule with the release of the second fragment of the hydrolysable protein and the initial form of the enzyme.
enzyme biological catalysis transamination
The discovery of the spatial structure of a number of enzymes by X-ray diffraction analysis provided a reliable basis for constructing rational schemes of their mechanism of action.
Establishing the mechanism of enzyme action is of key importance for revealing structural and functional relationships in a variety of biologically active systems.
Lysozyme is found in various tissues of animals and plants, it is found, in particular, in tear fluid and egg white. Lysozyme functions as an antibacterial agent by catalyzing the hydrolysis of the cell walls of a number of bacteria. This polysaccharide is formed by alternating N-acetylmuranoic acid (NAM) residues connected by a 1,4-glycosidic bond (polysaccharide chains are crosslinked by short peptide fragments).
The bacterial polysaccharide is a very complex insoluble compound; therefore, well hydrolysable oligosaccharides formed by NAG residues are often used as lysozyme substrates.
Chicken egg protein lysozyme is formed by a single polypeptide chain containing 129 amino acid residues; its molecular weight is 14,600. The high stability of the enzyme is ensured by the presence of four disulfide bridges.
Information about the active center and the type of catalytic process was obtained by D. Philips in 1965. based on X-ray diffraction studies of lysozyme and its complexes with inhibitors. The lysozyme molecule has the shape of an ellipsoid with axes 4.5*3*3 nm; between the two halves of the molecule is a "gap" in which the binding of oligosaccharides occurs. The walls of the gap are formed mainly by the side chains of non-polar amino acids, which ensure the binding of non-polar molecules of the substrate, and also include the side chains of polar amino acids, which are capable of forming hydrogen bonds with the acylamino and hydroxyl groups of the substrate. The size of the gap allows to accommodate an oligosaccharide molecule containing 6 monosaccharide residues. It is not possible to establish the nature of the binding of the substrate, for example, the hexasaccharide NAG 6 , by the method of X-ray diffraction analysis. At the same time, complexes of the enzyme with the trisaccharide inhibitor NAG 3 are stable and well studied. NAG 3 binds in a gap on the surface of the enzyme, forming hydrogen bonds and van der Waals contacts; at the same time, it fills only half of the gap, in which three more monosaccharide residues can bind. The non-reducing end (sugar A) is at the beginning of the gap, and the reducing end (sugar C) is in its central part; sugar residues A, B and C have a chair conformation. The construction of a model of the enzyme-substrate complex was based on the assumption that the same interactions are realized upon binding of the NAG 6 substrate as upon the binding of NAG 3 . In the enzyme model, three sugar residues (referred to as residues D, E, and F) were placed inside the gap; each subsequent sugar was attached in such a way that its conformation was the same (as far as possible) as that of the first three sugars. As part of the model complex, all sugar residues implement effective non-covalent interactions with side and peptide groups of amino acid residues that form a gap.
When identifying catalytic groups, it was natural to focus on those that are in the enzyme-substrate complex near the cleavable glycosidic bond and can serve as proton donors or acceptors. It turned out that on one side of the split bond, at a distance? 0.3 nm (from the oxygen of the glycosidic bond), the carboxyl group of Glu-35 is located, and on the other (at the same distance) the carboxyl group of Asp-52, their environment is very different. Glu-35 is surrounded by hydrophobic residues; it can be assumed that at the optimum pH of the enzyme, this group is in a non-ionized state. The environment of Asp-52 is pronounced polar; its carboxyl group participates as a hydrogen acceptor in a complex network of hydrogen bonds and probably functions in an ionized state.
The following scheme of the catalytic process during the hydrolysis of the oligosaccharide has been proposed. The non-ionized carboxyl group of Glu-35 acts as a proton donor, supplying it to the glycosidic oxygen atom between the C (1) atom of sugar D and the C (4) atom of sugar E (general acid catalysis stage); this results in the breaking of the glycosidic bond. As a result, the sugar residue D passes into the state of a carbocation with a positively charged carbon atom C (1) and takes on a half-chair conformation. The negative charge of the Asp-52 carboxylate group stabilizes the carbocation. The remainder of NAG 2 (sugars E+F) diffuses out of the active site region. Then a water molecule enters the reaction; its proton passes to Glu-35, and the OH - group to the C (1) atom of the D residue (the stage of basic catalysis). The NAG 4 residue (sugars A + B + C + D) leaves the region of the active center, and the enzyme returns to its original state.
Ribonuclease (RNase) of the bovine pancreas hydrolyzes internucleotide bonds in RNA near the pyrymylin units, which remain esterified at the 3 "position. The enzyme, along with other nucleases, is widely used in the analysis of the structure of RNA.
RNase is formed by one polypeptide chain containing 124 amino acid residues, and its molecular weight is 13,680; There are four disulfide bonds in the molecule. RNase is the first enzyme for which a primary structure has been established.
Based on the results of the study of ribonuclease renaturation, K. Afinsen for the first time clearly formulated the idea that the spatial structure of a protein is determined by its primary structure.
In 1958, F. Richards showed that, under certain conditions, subtilisin cleaves the peptide bond Ala-20 - Ser-21 in RNase. The resulting fragments were called S-peptide (residues 1-20) and S-protein (residues 21-124); due to non-covalent interactions, the fragments form a complex called RNase S. This complex has almost the full catalytic activity of the native enzyme; in isolated form, S-peptide and S-protein are inactive. Further, it was found that a synthetic peptide identical in sequence to the S-peptide fragment containing residues 1 to 13 restores the activity of the S-protein, but a shorter peptide containing residues 1 to 11 does not have this ability. The data obtained allowed us to conclude that the corresponding His-12 or Met-13 residues (or both of these residues) are included in the active site of the enzyme.
When studying the effect of pH on RNase activity, the important role of protein functional groups with pK 5.2 and 6.8 was elucidated; this suggested the participation of histidine residues in the catalytic process.
Upon carboxylation of RNase with iodoacetate at pH 5.5, i.e. under conditions under which the modification of histidine residues predominantly occurs, a complete loss of activity was observed; the modified enzyme contains 1 mol of carboxymethyl groups per 1 mol of protein. As a result, two monocarboxymethylene forms of the enzyme are formed. In one form, His-12 is carboxymethylated, and in the other, His-119. His-119 was predominantly modified.
These data suggested that His-12 and His-119 are in the active site and that modification of one of them prevents modification of the other.
As a result of X-ray diffraction studies, the spatial structure of RNase S and the complex of RNase S with inhibitors was elucidated. The molecule has the shape of a kidney, the active center is localized in the depression where the residues of His-12, His-119 and Lys-41 are located.
Hydrolysis occurs as a result of the conjugated action of His-12 and His-119 residues, which carry out acid-base catalysis. The diagram below shows the stages of the catalytic process:
1. The substrate is in the active site; His-12, His-119 and Lys-41 are located near the negatively charged phosphate.
2. As a result of the action of His-12 as a base that accepts a proton from the 2 "-OH group of ribose, and His-119 as an acid that donates a proton to the oxygen atom of the phosphate, an intermediate complex is formed first, and then a 2", 3 "-cyclic phosphate .
3. In place of the departed product, water enters, donating the proton of His-119, and OH to phosphate, at the same time the proton from His-12 passes to the oxygen atom of ribose, the second product is formed, and the enzyme returns to its original state.
Chymotrypsin is secreted in the form of a proenzyme - chymotrypsinogen by the pancreas of vertebrates; proenzyme activation occurs in the duodenum under the action of trypsin. The physiological function of chymotrypsin is the hydrolysis of proteins and polypeptides. Chymotrypsin attacks mainly peptide bonds formed by carboxyl residues of tyrosine, tryptophan, cenylalanine and methionanine. It also effectively hydrolyses the esters of the corresponding amino acids. The molecular weight of chymotrypsin is 25,000, the molecule contains 241 amino acid residues. Chymotrypsin is formed by three polypeptide chains linked by disulfide bridges.
The functional groups of the active site of chymotrypsin have been identified using irreversible inhibitors. The Ser-195 residue was modified with diisopropyl fluorophosphate and phenylmethylsulfofluoride, and the His-122 residue was modified with N-tosyl-L-phenylalanine-chloromethyl ketone. The two-stage process of chymotrypsin hydrolysis was discovered in the study of the kinetics of hydrolysis of p-nitrophenylacetate.
A characteristic feature of the process under consideration is the formation of a covalent intermediate, an acyl enzyme. The acylated catalytic group was identified as the residue Ser-195. The mechanism of catalysis carried out by the enzyme was proposed even before the establishment of the spatial structure of the protein, but was later refined. In particular, studies using 18 H 2 O made it possible to prove the formation of an acyl enzyme during the hydrolysis of peptides.
A three-dimensional structure with a resolution of 0.2 nm was established by D. Blow's X-ray diffraction analysis. in 1976 The molecule has the shape of an ellipsoid with axes 5.4*4*4 nm. The results of crystallographic studies confirmed the assumption that the Ser-195 and His-57 residues are close. The hydroxyl group of Ser-195 is located at a distance of ~0.3 nm orth of the nitrogen atom of the His-57 imidazole ring. The most interesting fact was that the nitrogen atom in position 1 of the ring is located at a distance of ~0.28 nm from the oxygen atom of the carboxyl group of the Asp-102 side chain and occupies a position favorable for the formation of a hydrogen bond.
It should be noted that chemical studies could not reveal the involvement of Asp-102 in the functioning of the active center, since this residue is embedded deep into the molecule.
It is currently believed that the three residues Asp-102, His-57 and Ser-195 form a charge transfer system that plays a critical role in the catalysis process. The functioning of the system ensures the effective participation of His-57 in catalysis as an acid-base catalyst and increases the reactivity of Ser-195 to the carboxyl carbon of the attacked bond.
The key element of catalysis is the proton transfer from Ser-195 to His-57. At the same time, the oxygen atom of serine attacks the carbonyl carbon atom of the substrate with the formation of first an intermediate tetrahedral compound (1), and then an acyl enzyme (2). The next step is deacylation. The water molecule enters the charge transfer system, and the OH ion simultaneously attacks the carbonyl carbon atom of the acyl group of the acyl enzyme. As in the acylation step, an intermediate tetrahedral compound (4) is formed. His-57 then donates a proton to the oxygen atom of Ser-195, releasing the acyl product; it diffuses into the solution, and the enzyme returns to its original state.
Carboxypeptidase A is secreted as a proenzyme by the pancreas of vertebrates. The formation of the active enzyme occurs in the small intestine with the participation of chymotrypsin. The enzyme sequentially cleaves off C-terminal amino acid residues from the peptide chain, i.e. is an exopeptidase.
Carboxypeptidase A is formed by a single polypeptide chain containing 307 amino acid residues; the molecular weight is 34,470. The amino acid sequence of the protein was established in 1969 by R. Bredshaw.
Elucidation of the mechanism of action of the enzyme was possible only after X-ray diffraction studies. The spatial structure of the enzyme and its complex with the Gly-Tyr dipeptide (substrate model) was established by W. Lipscomb. The enzyme molecule has the shape of an ellipsoid with axes 5.0*4.2*3.8 nm; the active center is located in a depression that passes into a deep non-polar pocket. A zinc ion is localized in the active center zone (its ligands are the side chains of Glu-72, His196, His-69 residues and a water molecule), as well as functional groups involved in substrate binding and catalysis - Arg-145, Glu-270 and Tyr-248.
A comparative analysis of the structures of the enzyme and its complex with Gly-Tyr yielded important information on the structure of the enzyme-substrate complex. In particular, it was found that during the formation of the complex, the hydroxyl group of Tyr-248 moves 1.2 nm relative to its position in the free enzyme (ie, approximately 1/3 of the molecule diameter).
According to the scheme of the catalytic process, the carboxylate group of Glu-270 activates a water molecule located in the reaction sphere, pulling a proton from it; the resulting OH- ion carries out a nucleophilic attack on the carbonyl carbon of the cleavable bond. At the same time, the hydroxyl group of Tyr-248, located near the nitrogen atom of the cleavable peptide bond, donates a proton to it. As a result, the attacked peptide bond is cleaved and the resulting products leave the active site zone. The diagram below illustrates the general basic catalysis.
Aspartate aminotransferase catalyzes the reversible transamination reaction.
The enzymatic transamination reaction was discovered by A.E. Braunstein and M.G. Kritzman in 1937 in the study of an enzyme preparation from the muscle of a pigeon. In subsequent studies, it was shown that transamination reactions are widespread in wildlife and play an important role in the conjugation of nitrogen and energy metabolism.
In 1945, it was found that pyridoxal-5 "-phosphate (PLP) is a coenzyme of aminotransferases. The AAT molecule is a dimer formed by identical subunits. In the cardiac muscle of the investigated vertebrates, there are two isoenzymes - cytoplasmic (cAAT0) and mitochondrial (mAAT) aminotransferases.
The primary structure of cAAT from cardiac muscle was established in 1972. Yu.A. Ovchinnikov and A.E. Brainstein. The polypeptide chain of a protein contains 412 amino acid residues; molecular weight is 46,000.
The general theory of pyridoxal catalysis was developed by A.E. Braunstein and M.M. Shemyakin in 1952-1953, and somewhat later - D.E. Metzler and E.E. Snell. According to this theory, the catalytic action of pyridoxal enzymes is due to the ability of the aldehyde group of pyridoxal phosphate to form aldimines (Schiff bases) when interacting with amines, including amino acids.
In the resulting phosphopyridoxyldeneamino acid, there is a system of conjugated double bonds, along which the displacement of electrons from the 6-carbon atom facilitates the breaking of the bonds formed by this atom.
Modern ideas about the mechanism of enzymatic transamination, developed by A.E. Braunstein and his collaborators are a development of the above theory. In the initial state, the aldehyde group of pyridoxal phosphate forms an aldimine bond with the e-amino group of the Lys-258 residue of the active center (I). Upon binding of the amino acid, a Michaelis complex (II) is formed, followed by an aldimine between pyridoxal phosphate and substrate (III). As a result of subsequent transformations through intermediate stages (IV) and (V), oxo acid (VI) is formed. This completes the first half-reaction of transamination. Repeating these same steps in the "reverse" direction with the new hydroxy acid constitutes the second half-reaction that completes the catalytic transamination cycle.
Myoglobin and hemoglobin
These two proteins are often referred to as respiratory enzymes. Their interaction with the substrate, oxygen, has been elucidated in detail, primarily on the basis of high-resolution X-ray diffraction analysis. The three-dimensional structure of myoglobin was determined by J. Kendrew in 1961, and the three-dimensional structure of hemoglobin - by M. Perutz in 1960.
The myoglobin molecule has a compact shape - 4.5 * 3.5 * 2.5 nm, the polypeptide chain forms 8 helical sections, denoted by letters from A to H. It is arranged in a specialized way around a large flat iron-containing heme ring. Heme is a complex of porphyrin with ferrous iron.
The polar heme propionic acid chains are located on the surface of the molecule, the rest of the heme is embedded in the globule. The connection of heme with the protein is carried out due to the coordination bond between the iron atom and the histidine atom, localized in the F helix; this is the so-called proximal histidine. Another important histidine residue, distal histidine, is localized in the heme pocket in the E helix; it is located on the opposite side of the iron atom at a greater distance than the proximal histidine. The region between the gene iron and the distal histidine in deoxymyoglobin is free, and the lipophilic O 2 molecule can bind to the heme iron, occupying the sixth coordination position. A unique feature of myoglobin, as well as hemoglobin, is their ability to reversibly bind O 2 without oxidizing heme Fe 2+ to Fe 3+ . This is possible because a low permittivity medium is created in the hydrophobic heme pocket from which water is displaced.
When O 2 is bound to an iron atom, the latter moves by about 0.06 nm and ends up in the plane of the porphyrin ring, i.e. in an energetically more favorable position. It is believed that this movement is due to the fact that the Fe 2+ ion in deoxymyoglobin is in a high-spin state and its radius is too large to fit in the plane of the heme porphyrin ring. When O 2 is bound, the Fe 2+ ion passes into a low-pin state and its radius decreases; now the Fe 2+ ion can move into the plane of the porphyrin ring.
Hemoglobin is the main component of red blood cells that delivers oxygen from the lungs to the tissues, and carbon dioxide from the tissues to the lungs. Hemoglobins of different types differ in the form of crystals, solubility, affinity for oxygen. This is due to differences in the amino acid sequence of proteins; the heme component is the same in hemoglobins of all vertebrate species and some invertebrates.
Human hemoglobin is a tetramer consisting of four subunits, two b-subunits and two b-subunits, each containing 141 and 146 amino acid residues, respectively. There is significant homology between the primary structures of the β and β subunits, and the conformation of their polypeptide chains is also similar.
The hemoglobin molecule has a spherical shape with a diameter of 5.5 nm. The four subunits are packed in a tetrahedral shape.
X-ray diffraction data showed that oxygenation of hemoglobin is accompanied by a number of changes. At low resolution, it was found that in this case the structure becomes more compact (the Fe atoms of the β-chains approach each other by about 0.6-0.7 nm), the subunits rotate relative to each other and the second-order axis by 10-15 o. The results of the study at high resolution indicate that especially significant changes occur in the region of 6v contacts.
To date, on the basis of X-ray diffraction studies and a number of other methodological approaches, significant progress has been made in elucidating the mechanism of action of enzymes with desired properties based on achievements in the field of genetic engineering. This opens up wide opportunities for testing the validity of modern ideas about the mechanism of enzyme action and creating a fundamental theory of enzymatic catal.
V 3 = K 3* [ F 0 ] * [ S] / km.
In this equation K 3 , F 0 ], km - constants and can be replaced by a new constant K*. Thus, at a low substrate concentration, the reaction rate is directly proportional to this concentration.
V 3 = K* * [ S].
This dependence corresponds to the first section of graph 2.
2. The dependence of the rate on the concentration of the enzyme manifests itself at a high concentration of the substrate.
S?Km.
In this case, Km can be neglected and the equation becomes:
V 3 = K 3* (([ F 0 ] * [ S]) / [ S]) = K 3* [ F 0 ] = V max.
Thus, at a high substrate concentration, the reaction rate is determined by the enzyme concentration and reaches its maximum value
V 3 = K 3 [ F 0 ] = V max. ( third section of graph 2).
3. Allows you to determine the numerical value of Km under the condition V 3 = V max /2. In this case, the equation takes the form:
V max /2 = ((V max * [S]) / Km+ [S]), whence it follows that Km= [S]
Thus, Km is numerically equal to the concentration of the substrate at a reaction rate equal to half the maximum. Km is a very important characteristic of an enzyme, it is measured in moles (10 -2 - 10 -6 mol) and characterizes the specificity of the enzyme: the lower the Km, the higher the specificity of the enzyme.
Graphic definition constants Michaelis.
It is more convenient to use a graph representing a straight line.
Such a graph is proposed by Lineweaver - Burke (plot of double reciprocals), which corresponds to the inverse Michaelis - Menten equation
Dependence of the rate of enzymatic reactions on the presence of activators and inhibitors
Activators - substances that increase the rate of enzymatic reactions. There are specific activators that increase the activity of one enzyme (HCl - pepsinogen activator) and non-specific activators that increase the activity of a number of enzymes (Mg ions - activators of hexokinase, K, Na - ATPase and other enzymes). Metal ions, metabolites, nucleotides can serve as activators.
The mechanism of action of activators
1. Completion of the active center of the enzyme, as a result of which the interaction of the enzyme with the substrate is facilitated. This mechanism is mainly possessed by metal ions.
2. The allosteric activator interacts with the allosteric site (subunit) of the enzyme, through its changes indirectly changes the structure of the active center and increases the activity of the enzyme. The metabolites of enzymatic reactions, ATP, have an allosteric effect.
3. The allosteric mechanism can be combined with a change in the oligomerism of the enzyme. Under the action of the activator, several subunits are combined into an oligomeric form, which dramatically increases the activity of the enzyme. For example, isocitrate is an activator of the enzyme acetyl-CoA carboxylase.
4. Phospholylation - dephosphorylation of enzymes refers to the reversible modification of enzymes. The addition of H 3 RO 4 most often sharply increases the activity of the enzyme. For example, two inactive dimers of the phosphorylase enzyme combine with four ATP molecules to form the active tetrameric phosphorylated form of the enzyme. The phosphorylation of enzymes can be combined with a change in their oligomericity. In some cases, phosphorylation of the enzyme, on the contrary, reduces its activity (for example, phosphorylation of the enzyme glycogen synthetase)
5. Partial proteolysis (irreversible modification). With this mechanism, a fragment of the molecule is cleaved off from the inactive form of the enzyme (proenzyme), blocking the active center of the enzyme. For example, inactive pepsinogen is converted to active pepsin by HCL.
Inhibitors - substances that reduce enzyme activity.
By specificity distinguish between specific and non-specific inhibitors
By reversibility effect distinguish between reversible and irreversible inhibitors.
By place actions there are inhibitors that act on the active center and outside the active center.
By mechanism actions distinguish between competitive and non-competitive inhibitors.
Competitive inhibition .
Inhibitors of this type have a structure close to that of the substrate. Because of this, inhibitors and the substrate compete for the binding of the active site of the enzyme. Competitive inhibition is reversible inhibition The effect of a competitive inhibitor can be reduced by increasing the concentration of the reaction substrate
An example of competitive inhibition is the inhibition of the activity of succinate dehydrogenase, which catalyzes the oxidation of dicarboxylic succinic acid, by dicarboxylic malonic acid, similar in structure to succinic acid.
The principle of competitive inhibition is widely used in the development of drugs. For example, sulfanilamide preparations have a structure close to the structure of para-aminobenzoic acid, which is necessary for the growth of microorganisms. Sulfonamides block the enzymes of microorganisms necessary for the absorption of para-aminobenzoic acid. Some anticancer drugs are analogues of nitrogenous bases and thus inhibit the synthesis of nucleic acids (fluorouracil).
Graphically, competitive inhibition has the form:
Non-competitive inhibition .
Non-competitive inhibitors are not structurally similar to reaction substrates and therefore cannot be displaced at high substrate concentrations. There are several options for the action of non-competitive inhibitors:
1. Blocking of the functional group of the active center of the enzyme and, as a result, a decrease in activity. For example, the activity of SH - groups can bind thiol poisons reversibly (salts of metals, mercury, lead) and irreversibly (monioiodoacetate). The inhibition effect of thiol inhibitors can be reduced by the introduction of additional substances rich in SH groups (for example, unithiol). There are and are used serine inhibitors that block the OH - groups of the active center of enzymes. Organic phosphofluorine-containing substances have this effect. These substances can, in particular, inhibit OH groups in the enzyme acetylcholinesterase, which destroys the neurotransmitter acetylcholine.
2. Blocking of metal ions that are part of the active center of enzymes. For example, cyanides block iron atoms, EDTA (ethylenediaminetetraacetate) blocks Ca, Mg ions.
3. An allosteric inhibitor interacts with the allosteric site, indirectly through it according to the principle of cooperativity, changing the structure and activity of the catalytic site. Graphically, non-competitive inhibition has the form:
The maximum reaction rate in non-competitive inhibition cannot be achieved by increasing the substrate concentration.
Regulation of enzyme activity during metabolism
Adaptation of the body to changing conditions (diet, environmental impacts, etc.) is possible due to a change in the activity of enzymes. There are several possibilities for regulating the rate of enzymatic reactions in the body:
1. Change in the rate of enzyme synthesis (this mechanism requires a long period of time).
2. Increasing the availability of the substrate and the enzyme by changing the permeability of cell membranes.
3. Change in the activity of enzymes already present in cells and tissues. This mechanism is carried out at high speed and is reversible.
In multistage enzymatic processes, regulatory, key enzymes are isolated that limit the overall rate of the process. Most often, these are enzymes of the initial and final stages of the process. Changes in the activity of key enzymes occur through various mechanisms.
1. Allosteric mechanism:
2. Change in enzyme oligomerism:
Monomers are inactive - oligomers are active
3. Phospholylation - dephosphorylation:
Enzyme (inactive) + H 3 RO 4 - phosphorylated active enzyme.
Autoregulatory mechanism is widespread in cells. The autoregulatory mechanism is, in particular, retroinhibition, in which the products of the enzymatic process inhibit the enzymes of the initial stages. For example, high concentrations of purine and pyrimidine nucleotides inhibit the initial ones at the stage of their synthesis.
Sometimes the initial substrates activate the final enzymes, in the scheme: substrate A activates F 3 . For example, the active form of glucose (glucose-6-phosphate) activates the final enzyme in the synthesis of glycogen from glucose (glycogen synthetase).
Structural organization of enzymes in a cell
The coherence of metabolic processes in the body is possible due to the structural dissociation of enzymes in cells. Individual enzymes are located in certain intracellular structures - compartmentalization . For example, the enzyme potassium, sodium ATPase, is active in the plasma membrane. In mitochondria, enzymes of oxidative reactions (succinate dehydrogenase, cytochrome oxidase) are active. Enzymes for the synthesis of nucleic acids (DNA polymerase) are active in the nucleus. In lysosomes, enzymes for the breakdown of various substances (RNase, phosphatase, and others) are active.
Enzymes that are most active in a given cell structure are called indicator or marker enzymes. Their definition in clinical practice reflects the depth of structural tissue damage. Some enzymes combine into polyenzymatic complexes, for example, the pyruvate dehydrogenase complex (PDC), which oxidizes pyruvic acid.
PrinciplesdetectionAndquantitativedefinitionsenzymes:
The detection of enzymes is based on their high specificity. Enzymes are detected by the action they produce, i.e. upon the occurrence of the reaction catalyzed by the enzyme. For example, amylase is detected by the breakdown of starch to glucose.
The criteria for the occurrence of an enzymatic reaction can be:
disappearance of the reaction substrate
appearance of reaction products
change in the optical properties of the coenzyme.
Quantification of enzymes
Since the concentration of enzymes in cells is very low, it is not their true concentration that is determined, but the amount of the enzyme is judged indirectly, by the activity of the enzyme.
Enzyme activity is assessed by the rate of the enzymatic reaction occurring under optimal conditions (optimal temperature, pH, excessively high concentration of the substrate). Under these conditions, the reaction rate is directly proportional to the concentration of the enzyme (V= K 3 ).
Units activity ( quantity ) enzyme
In clinical practice, several units of enzyme activity are used.
1. International unit - the amount of enzyme that catalyzes the conversion of 1 micromol of substrate per minute at a temperature of 25 0 C.
2. Catal (in the SI system) - the amount of enzyme that catalyzes the transformation of 1 mole of the substrate per second.
3. Specific activity - the ratio of enzyme activity to the mass of enzyme protein.
4. The molecular activity of an enzyme shows how many substrate molecules are converted under the action of 1 enzyme molecule.
Clinical fermentology
The use of information about enzymes in medical practice is a branch of medical enzymology. It includes 3 sections:
1. Enzymodiagnostics
2. Enzymopotology
3. Enzyme therapy
Enzymodiagnostics - a section that studies the possibilities of studying the activity of enzymes for diagnosing diseases. To assess the damage to individual tissues, organ-specific enzymes, isoenzymes are used.
In pediatric practice, when conducting enzyme diagnostics, it is necessary to take into account children's characteristics. In children, the activity of some enzymes is higher than in adults. For example, high LDH activity reflects the predominance of anaerobic processes in the early postnatal period. The content of transaminases in the blood plasma of children is increased as a result of increased vascular tissue permeability. The activity of glucose-6-phosphate dehydrogenase is increased as a result of increased breakdown of erythrocytes. The activity of other enzymes, on the contrary, is lower than in adults. For example, the activity of pepsin, pancreatic enzymes (lipase, amylase) is reduced due to the immaturity of secretory cells.
With age, redistribution of individual isoenzymes is possible. So, in children LDH 3 (more anaerobic form) prevails, and in adults LDH 2 (more aerobic form).
Enzymopathology - a branch of fermentology that studies diseases, the leading mechanism of development of which is a violation of the activity of enzymes. These include metabolic disorders of carbohydrates (galactosemia, glycogenosis, mucopolysaccharidoses), amino acids (phenylketonuria, cystinuria), nucleotides (orotataciduria), porphyrins (porphyrias).
enzyme therapy - branch of fermentology that studies the use of enzymes, coenzymes, activators, inhibitors for therapeutic purposes. Enzymes can be used with a substitution purpose (pepsin, pancreatic enzymes), with a lytic purpose to remove necrotic masses, blood clots, to thin viscous exudates.
Literature
1. Avdeeva, L.V. Biochemistry: Textbook / L.V. Avdeeva, T.L. Aleinikova, L.E. Andrianova; Under the editorship of E.S. Severin. - M.: GEOTAR-MED, 2013. - 768 p.
2. Auerman, T.L. Fundamentals of Biochemistry: Textbook / T.L. Auerman, T.G. Generalova, G.M. Suslyanok. - M.: NITs INFRA-M, 2013. - 400 p.
3. Bazarnova Yu.G. Biochemical bases of processing and storage of raw materials of animal origin: Textbook / Yu.G. Bazarnova, T.E. Burova, V.I. Marchenko. - St. Petersburg: Ave. Science, 2011. - 192 p.
4. Baishev, I.M. Biochemistry. Test questions: Textbook / D.M. Zubairov, I.M. Baishev, R.F. Baykeev; Under the editorship of D.M. Zubairov. - M.: GEOTAR-Media, 2008. - 960 p.
5. Bokut, S.B. Biochemistry of phylogenesis and ontogenesis: Textbook / A.A. Chirkin, E.O. Danchenko, S.B. Bokut; Under total ed.A. A. Chirkin. - M.: NITs INFRA-M, Nov. knowledge, 2012. - 288 p.
6. Gidranovich, V.I. Biochemistry: Textbook / V.I. Gidranovich, A.V. Gidranovich. - Minsk: TetraSystems, 2012. - 528 p.
7. Goloshchapov, A.P. Genetic and biochemical aspects of human adaptation to the conditions of a city with a developed chemical industry / A.P. Goloshchapov. - M.: KMK, 2012. - 103 p.
8. Gunkova, P.I. Biochemistry of milk and dairy products / K.K. Gorbatova, P.I. Gunkov; Under total ed.K. K. Gorbatov. - St. Petersburg: GIORD, 2010. - 336 p.
9. Dimitriev, A.D. Biochemistry: Textbook / A.D. Dimitriev, E.D. Ambrosiev. - M.: Dashkov i K, 2013. - 168 p.
10. Ershov, Yu.A. General biochemistry and sport: Textbook / Yu.A. Ershov. - M.: MGU, 2010. - 368 p.
11. Ershov, Yu.A. Fundamentals of Biochemistry for Engineers: Textbook / Yu.A. Ershov, N.I. Zaitsev; Under the editorship of S.I. Schukin. - M.: MSTU im. Bauman, 2010. - 359 p.
12. Kamyshnikov, V.S. Handbook of clinical and biochemical laboratory diagnostics: In 2 volumes. In 2 volumes. Handbook of clinical and biochemical laboratory diagnostics: In 2 volumes / V.S. Kamyshnikov. - Minsk: Belarus, 2012. - 958 p.
13. Klopov, M.I. Biologically active substances in physiological and biochemical processes in the animal body: Textbook / M.I. Klopov, V.I. Maksimov. - St. Petersburg: Lan, 2012. - 448 p.
14. Mikhailov, S.S. Sports biochemistry: A textbook for universities and colleges of physical culture / S.S. Mikhailov. - M.: Sov. sport, 2012. - 348 p.
15. Repnikov, B.T. Commodity science and biochemistry of fish products: Textbook / B.T. Repnikov. - M.: Dashkov i K, 2013. - 220 p.
16. Rogozhin, V.V. Biochemistry of milk and meat: Textbook / V.V. Rogozhin. - St. Petersburg: GIORD, 2012. - 456 p.
17. Rogozhin, V.V. Plant Biochemistry: Textbook / V.V. Rogozhin. - St. Petersburg: GIORD, 2012. - 432 p.
18. Rogozhin, V.V. Workshop on plant physiology and biochemistry: Textbook / V.V. Rogozhin, T.V. Rogozhin. - St. Petersburg: GIORD, 2013. - 352 p.
19. Taganovich, A.D. Pathological biochemistry: Monograph / A.D. Taganovich. - M.: BINOM, 2013. - 448 p.
20. Filippovich, Yu.B. Biochemical foundations of human life: Textbook for university students / Yu.B. Filippovich, A.S. Konichev, G.A. Sevastyanova, N.M. Kutuzov. - M.: VLADOS, 2005. - 407 p.
21. Shcherbakov, V.G. Biochemistry and commodity science of oil raw materials / V.G. Shcherbakov, V.G. Lobanov. - M.: KolosS, 2012. - 392 p.
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ChapterIV.3.
Enzymes
Metabolism in the body can be defined as the totality of all chemical transformations undergone by compounds coming from outside. 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 proceed in the body at an extremely high rate only in the presence of catalysts. All biological catalysts are substances of a 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 transformations. The 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 wide variety of reactions in the body. So, quite simple from the point of view of traditional chemistry, the reaction of splitting off 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 the pH of the blood. Thanks to the catalytic action of enzymes in the body, it becomes possible to carry out such reactions that would go hundreds and thousands of times slower without a catalyst.
Enzyme Properties
1. Influence on the rate of a chemical reaction: enzymes increase the rate of a chemical reaction, but they themselves are not consumed.
The reaction rate is the change in the concentration of the reaction components per unit time. If it goes in the forward direction, then it is proportional to the concentration of the reactants; if it goes 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 in the presence of enzymes comes faster.
2. The specificity of the action of enzymes. In the cells of the body, 2-3 thousand reactions take place, each of which is catalyzed by a certain enzyme. The specificity of the action of an enzyme is the ability to accelerate the course of one particular reaction without affecting the rate of others, even very similar ones.
Distinguish:
Absolute– when F catalyzes only one specific reaction ( arginase- breakdown of arginine)
Relative(group special) - F catalyzes a certain class of reactions (eg 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. The activity of enzymes is the ability to accelerate the reaction rate to varying degrees. Activity is expressed in:
1) International units of activity - (IU) the amount of the enzyme catalyzing the conversion of 1 μM of the substrate in 1 min.
2) Katalakh (cat) - the amount of catalyst (enzyme) capable of converting 1 mol of substrate in 1 s.
3) Specific activity - the number of units of activity (any of the above) in the test sample to the total mass of protein in this sample.
4) Less often, molar activity is used - the number of substrate molecules converted by one enzyme molecule per minute.
activity depends on temperature . This or that enzyme shows the greatest activity at an optimum temperature. For F of a living organism, this value is within +37.0 - +39.0° C, depending on the type of animal. With a decrease in temperature, 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. In case of temperature increase above +40 - +50° With the enzyme molecule, which is a protein, undergoes a process of denaturation. At the same time, the rate of the chemical reaction drops noticeably (Fig. 4.3.1.).
Enzyme activity also depends on medium pH . For most of them, there is a certain optimal pH value at which their activity is maximum. Since the cell contains hundreds of enzymes and each of them has its own opt pH limits, the change in pH is 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 opt of which lies in the range of 7.0 - 7.2, a product is formed, which is an acid. In this case, the pH value shifts to the region of 5.5 - 6.0. The activity of the enzyme sharply decreases, the rate of product formation slows down, but 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).
The chemical nature of enzymes. The structure of the enzyme. Active and allosteric centers
All enzymes are proteins with a molecular weight of 15,000 to several million Da. According to the chemical structure, they are simple enzymes (consist only of AA) and complex enzymes (have a non-protein part or a prosthetic group). The protein portion is called apoenzyme, and non-protein, if it is covalently linked to an apoenzyme, then 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 a 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 catalysis of the 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 when the protein molecule is twisted into a tertiary structure. Separate sections of amino acids approach each other, forming a certain configuration of the active center. An important structural feature of the active center is that its surface is complementary to the surface of the substrate; AA residues of this zone of the enzyme are able to enter into chemical interaction with certain groups of the substrate. It can be imagined that the active site of the enzyme matches the structure of the substrate like a key and a lock.
IN active center two zones are distinguished: binding center, responsible for the attachment of the substrate, and catalytic center responsible for the chemical transformation of the substrate. The composition of the catalytic center of most enzymes includes such AAs as Ser, Cys, His, Tyr, Lys. Complex enzymes in the catalytic center have a cofactor or coenzyme.
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.
The mechanism of action of enzymes
The act of catalysis consists of three successive stages.
1. Formation of an enzyme-substrate complex during interaction through the active center.
2. The binding of the substrate occurs at several points of the active center, which leads to a change in the structure of the substrate, its deformation due to a change in the bond energy in the molecule. This is the second stage and is called substrate activation. When this occurs, a certain chemical modification of the substrate and its transformation into a new product or products.
3. As a result of such a transformation, the new substance (product) loses its ability to be retained in the active center of the enzyme and the enzyme-substrate, or rather, the enzyme-product complex, dissociates (disintegrates).
Types of catalytic reactions:
A + E \u003d AE \u003d BE \u003d E + B
A + B + E \u003d AE + B \u003d ABE \u003d AB + E
AB + E \u003d ABE \u003d A + B + E, where E is an 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 are distinguished inhibitors - slowing down the rate of reaction and activators - accelerating the enzymatic reaction.
Depending on the mechanism of inhibition of the reaction, 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 with a lock (or almost coincides). The degree of this similarity may even be higher than with the substrate.
If A + E \u003d AE \u003d BE \u003d E + B, then I + E \u003d 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 chemicals of endogenous and exogenous origin (i.e., 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, maybe. they inhibit key metabolic reactions of microorganisms (sulfonamides) and tumor cells. But with an excess of the substrate and a low concentration of a competitive inhibitor, its action is canceled.
The second type of inhibitors is non-competitive. They interact with the enzyme outside the active site, and an excess of 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.
There are also reversible and irreversible inhibition. Irreversible inhibitors inactivate the enzyme by forming a chemical bond with its AA or other structural components. Usually this is a covalent bond with one of the sites of the active center. Such a complex practically does not dissociate under physiological conditions. In another case, the inhibitor disrupts the conformational structure of the enzyme molecule - causing its denaturation.
The action of reversible inhibitors can be removed by an excess of the substrate or by the action 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 effects,
2) form a complex with the substrate, which more actively binds to the active center of F,
3) interacting with an enzyme having a quaternary structure, they separate its subunits and thereby open 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. So 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 peculiar only to 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 the 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 related stages of metabolism occur.
Many enzymes are formed in cells and secreted into the anatomical cavities in an inactive state - these are proenzymes. Often in the form of proenzymes, proteolytic enzymes (break down proteins) are formed. Then, under the influence of pH or other enzymes and substrates, their chemical modification occurs and the active center becomes available 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. the name of the substrate with which it interacts
2. the nature of the catalyzed reaction
3. the 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 must be classified. Currently, an international classification of enzymes has been adopted, which is based on the type of catalyzed reaction. There are 6 classes, which in turn are divided into a number of subclasses (in this book they are presented only selectively):
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 B 2, B 5. 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 cleaved off by the enzyme, turning into the reduced form (NADH, NADPH, FADH) and transfer it to another enzyme-substrate complex, where it is given away.
1.2. Oxidase - catalyzes the transfer of hydrogen to oxygen with the formation of water or H 2 O 2. F. Cytochromoxysdase respiratory chain.
RH + NAD H + O 2 = ROH + NAD + H 2 O
1.3. Monooxidases - cytochrome P450. According to its structure, both hemo- and flavoprotein. It hydroxylates lipophilic xenobiotics (by the mechanism described above).
1.4. PeroxidasesAnd catalase- catalyze the decomposition of hydrogen peroxide, which is formed during metabolic reactions.
1.5. Oxygenases - catalyze the reactions of oxygen addition to the substrate.
2. Transferases - catalyze the transfer of various radicals from the donor molecule to the 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 (acyl group transfer - R-C=O).
Example: synthesis of the neurotransmitter acetylcholine (see chapter "Protein metabolism").
2.3. Hexosyl transferases catalyze the transfer of glycosyl residues.
Example: the splitting of a glucose molecule from glycogen under the action of phosphorylase.
2.4. Aminotransferases - transfer of amino groups
R 1- CO - R 2 + R 1 - CH - NH 3 - R 2 \u003d R 1 - CH - NH 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. Phosphotransferesis (kinase) - catalyze the transfer of a phosphoric acid residue. In most cases, ATP is the phosphate donor. Enzymes of this class are mainly involved in the process of glucose breakdown.
Example: Hexo (gluco) kinase.
3. Hydrolases - catalyze hydrolysis reactions, i.e. splitting of substances with addition at the place of breaking the bond of water. This class includes mainly digestive enzymes, they are one-component (do not contain a non-protein part)
R1-R2 + H 2 O \u003d R1H + R2OH
3.1.
Esterases - break down essential bonds. This is a large subclass of enzymes that catalyze the hydrolysis of thiol esters, phosphoesters.
Example: NH 2 ).
Example: arginase(urea cycle).
4. Liases - catalyze the reactions of cleavage of molecules without the addition of 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 commonly referred to as 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 the synthesis of more complex substances from simple ones. Such reactions proceed with the expenditure of ATP energy. Synthetase is added to the name of such enzymes.
LITERATURE TO THE CHAPTER IV.3.
1. Byshevsky A. Sh., Tersenov O. A. Biochemistry for a doctor // Ekaterinburg: Ural worker, 1994, 384 p.;
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.: Prosveschenie, 1982, 311 pp.;
4. Lehninger A. Biochemistry. Molecular bases of the structure and functions of the cell // M.: Mir, 1974, 956 p.;
5. Pustovalova L.M. Workshop on biochemistry // Rostov-on-Don: Phoenix, 1999, 540 p.
The mechanism of action of enzymes
The mechanism of action of enzymes can be considered from two positions: from the point of view of changes in the energy of chemical reactions and from the point of view of events in the active center.
A. Energy changes in chemical reactions
Any chemical reactions proceed, obeying two basic laws of thermodynamics: the law of conservation of energy and the law of entropy. According to these laws, the total energy of a chemical system and its environment remains constant, while the chemical system tends to reduce order (increase entropy). To understand the energy of a chemical reaction, it is not enough to know the energy balance of the reactants entering and exiting the reaction; it is necessary to take into account the energy changes in the course of a given chemical reaction and the role of enzymes in the dynamics of this process. Consider the decomposition reaction of carbonic acid:
H 2 CO 3 > H 2 0 + C0 2.
Carbonic acid is weak; the reaction of its decomposition will proceed under normal conditions, if the molecules of carbonic acid have an energy exceeding a certain level, called the activation energy E a (Fig. 2-10).
The activation energy is the additional amount of kinetic energy required by the molecules of a substance for them to react.
When this energy barrier is reached, changes occur in the molecule that cause the redistribution of chemical bonds and the formation of new compounds. Molecules with E a are said to be in a transition state. The energy difference between the initial reagent H 2 CO 3 and the final compounds H 2 O and CO 2 is called the change in free
![](https://i1.wp.com/studwood.ru/imag_/43/155548/image002.jpg)
Rice. 2-10. Change in free energy during the decomposition of carbonic acid.
Reaction energies DG. H 2 O and CO 2 molecules are more stable substances than H 2 CO 3 , i.e. have less energy and practically do not react under normal conditions. The released energy as a result of this reaction is dissipated in the form of heat into the environment.
The more molecules have an energy exceeding the level of E a, the higher the rate of a chemical reaction. The rate of a chemical reaction can be increased by heating. This increases the energy of the reacting molecules. However, high temperatures are detrimental to living organisms, so enzymes are used in the cell to speed up chemical reactions. Enzymes provide a high rate of reactions under optimal conditions existing in the cell, by lowering the level of Ea. Thus, enzymes lower the height of the energy barrier, as a result, the number of reactive molecules increases, therefore, the reaction rate increases.
In the mechanism of enzymatic catalysis, the formation of unstable intermediates is of decisive importance - the enzyme-substrate complex ES, which undergoes transformation into an unstable transition complex EP, which almost instantly decomposes into a free enzyme and a reaction product.
Thus, biological catalysts (enzymes) do not change the free energy
substrates and products and therefore do not change the equilibrium of the reaction (Fig. 2-11).
The enzyme, acting as a catalyst for a chemical reaction, obeys the general laws of catalysis and has all the properties characteristic of non-biological catalysts, however, it also has distinctive properties associated with the structural features of enzymes.
Enzymes are similar to non-biological catalysts in that:
- Enzymes catalyze energetically possible reactions;
- the energy of a chemical system remains constant;
- during catalysis, the direction of the reaction does not change;
- Enzymes are not consumed during the reaction.
The differences between enzymes and non-biological catalysts are that:
- · the rate of enzymatic reactions is higher than the reactions catalyzed by non-protein catalysts;
- Enzymes have high specificity;
- The enzymatic reaction takes place in the cell, i.e. at a temperature of 37 °C, constant atmospheric pressure and physiological pH value;
- The rate of the enzymatic reaction can be controlled.
1. Formation of the enzyme-substrate complex
The fact that enzymes have high specificity made it possible in 1890 to put forward a hypothesis according to which the active center of the enzyme is complementary to the substrate, i.e. corresponds to it as "the key to the lock". After the interaction of the substrate ("key") with the active center ("lock"), chemical transformations of the substrate into the product occur. The active center was considered as a stable, rigidly determined structure.
In 1959, another variant of the "key-lock" hypothesis was proposed to explain the events in the active site of the enzyme. According to this hypothesis, the active center is a flexible structure
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Rice. 2-11. The change in free energy during a chemical reaction, uncatalyzed and catalyzed by enzymes.
The enzyme lowers the activation energy E a, i.e. reduces the height of the energy barrier, as a result, the proportion of reactive molecules increases, therefore, the reaction rate increases with respect to the substrate. The substrate, interacting with the active site of the enzyme, causes a change in its conformation, leading to the formation of an enzyme-substrate complex that is favorable for chemical modifications of the substrate. In this case, the substrate molecule also changes its conformation, which ensures a higher efficiency of the enzymatic reaction. This "hypothesis of induced fit" subsequently received experimental confirmation.
2. Sequence of events during enzymatic catalysis
The process of enzymatic catalysis can be conditionally divided into the following stages (Fig. 2-12). substrate catalysis chemical reaction
The first, second, and fourth stages of catalysis are short and depend on the substrate concentration (for the first stage) and ligand binding constants in the active site of the enzyme (for the first and third stages). Changes in the energy of the chemical reaction at these stages are insignificant.
The third stage is the slowest; its duration depends on the activation energy of the chemical reaction. At this stage, bonds are broken in the substrate molecule, new bonds are formed, and the product molecule is formed.
3. Role of the active site in enzymatic catalysis
As a result of research, it was shown that the enzyme molecule, as a rule, is many times larger than the substrate molecule undergoing chemical transformation by this enzyme. Only a small part of the enzyme molecule comes into contact with the substrate, usually from 5 to 10 amino acid residues, which form the active site of the enzyme. The role of the remaining amino acid residues is to ensure the correct conformation of the enzyme molecule for the optimal course of the chemical reaction.
The active site at all stages of enzymatic catalysis cannot be considered as a passive site for substrate binding. It is a complex molecular "machine" that uses a variety of chemical mechanisms to promote the transformation of a substrate into a product.
In the active center of the enzyme, the substrates are arranged in such a way that the functional groups of the substrates participating in the reaction are in close proximity to each other. This property of the active center is called the effect of approach and orientation of the reactants. Such an ordered arrangement of substrates causes a decrease in entropy and, as a consequence, a decrease in the activation energy (E a), which determines the catalytic efficiency of enzymes.
The active center of the enzyme also contributes to the destabilization of interatomic bonds in the substrate molecule, which facilitates the course of a chemical reaction and the formation of products. This property of the active site is called the substrate deformation effect (Fig. 2-12).
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Rice. 2-12. Stages of enzymatic catalysis.
I - stage of approach and orientation of the substrate relative to the active center of the enzyme; II - the formation of an enzyme-substrate complex (ES) as a result of induced compliance; III - deformation of the substrate and the formation of an unstable enzyme-product complex (EP); IV- disintegration of the complex (EP) with the release of reaction products from the active center of the enzyme and the release of the enzyme.
B. Molecular mechanisms of enzymatic catalysis
The mechanisms of enzymatic catalysis are determined by the role of the functional groups of the active center of the enzyme in the chemical reaction of the transformation of the substrate into the product. There are 2 main mechanisms of enzymatic catalysis: acid-base catalysis and covalent catalysis.
1. Acid-base catalysis
The concept of acid-base catalysis explains enzymatic activity by the participation of acid groups (proton donors) and/or basic groups (proton acceptors) in a chemical reaction. Acid-base catalysis is a common phenomenon. The amino acid residues that make up the active center have functional groups that exhibit the properties of both acids and bases.
The amino acids involved in acid-base catalysis primarily include Cys, Tyr, Ser, Lys, Glu, Asp, and His. The radicals of these amino acids in the protonated form are acids (proton donors), in the deprotonated form they are bases (proton acceptors). Due to this property of functional groups of the active site, enzymes become unique biological catalysts, in contrast to non-biological catalysts that can exhibit either acidic or basic properties.
An example of acid-base catalysis, in which Zn 2+ ions are cofactors, and the NAD + molecule is used as a coenzyme, is the liver alcohol dehydrogenase enzyme, which catalyzes the alcohol oxidation reaction (Fig. 2-13):
C 2 H 5 OH + NAD + > CH 3 -SON + NADH + H
2. Covalent catalysis
Covalent catalysis is based on the attack of nucleophilic (negatively charged) or electrophilic (positively charged) groups of the active center of the enzyme by substrate molecules with the formation of a covalent bond between the substrate and the coenzyme or the functional group of the amino acid residue (usually one) of the active center of the enzyme.
The action of serine proteases such as trypsin, chymotrypsin and thrombin is an example of the mechanism of covalent catalysis, when a covalent bond is formed between the substrate and the serine amino acid residue of the active site of the enzyme. The term "serine proteases" is due to the fact that the amino acid residue of serine is part of the active center of all these enzymes and is directly involved in catalysis. Let us consider the mechanism of covalent catalysis using the example of chymotrypsin, which hydrolyzes peptide bonds during protein digestion in the duodenum (see Section 9). The substrates of chymotrypsin are peptides containing amino acids with aromatic and cyclic
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Rice. 2-13. The mechanism of acid-base catalysis on the example of liver alcohol dehydrogenase.
I - an ethanol molecule has a binding center that provides hydrophobic interaction between the active center and the methyl group of alcohol; II - a positively charged zinc atom contributes to the elimination of a proton from the alcohol group of ethanol with the formation of a negatively charged oxygen atom. The negative charge is redistributed between the oxygen atom and the neighboring hydrogen atom, which is then transferred in the form of hydrithione to the fourth carbon atom of the NAD+ coenzyme nicotinamide; III - as a result, the reduced form of NADH and acetaldehyde are formed.
Hydrophobic radicals (Phen, Tyr, Tri), which indicates the participation of hydrophobic forces in the formation of the enzyme-substrate complex. The mechanism of covalent catalysis of chymotrypsin is considered in fig. 2-14.
The radicals Asp 102, Gis 57, and Ser 195 are directly involved in the act of catalysis. Due to the nucleophilic attack of the peptide bond of the substrate, this bond is broken with the formation of a covalently modified serine - acyl-chymotrypsin. Another peptide fragment is released as a result of breaking the hydrogen bond between the peptide fragment and His 57 of the chymotrypsin active site. The final step in the hydrolysis of the peptide bond of proteins is the deacylation of chymotrypsin in the presence of a water molecule with the release of the second fragment of the hydrolysable protein and the initial form of the enzyme.
enzyme biological catalysis transamination
The discovery of the spatial structure of a number of enzymes by X-ray diffraction analysis provided a reliable basis for constructing rational schemes of their mechanism of action.
Establishing the mechanism of enzyme action is of key importance for revealing structural and functional relationships in a variety of biologically active systems.
Lysozyme is found in various tissues of animals and plants, it is found, in particular, in tear fluid and egg white. Lysozyme functions as an antibacterial agent by catalyzing the hydrolysis of the cell walls of a number of bacteria. This polysaccharide is formed by alternating N-acetylmuranoic acid (NAM) residues connected by a 1,4-glycosidic bond (polysaccharide chains are crosslinked by short peptide fragments).
The bacterial polysaccharide is a very complex insoluble compound; therefore, well hydrolysable oligosaccharides formed by NAG residues are often used as lysozyme substrates.
Chicken egg protein lysozyme is formed by a single polypeptide chain containing 129 amino acid residues; its molecular weight is 14,600. The high stability of the enzyme is ensured by the presence of four disulfide bridges.
Information about the active center and the type of catalytic process was obtained by D. Philips in 1965. based on X-ray diffraction studies of lysozyme and its complexes with inhibitors. The lysozyme molecule has the shape of an ellipsoid with axes 4.5*3*3 nm; between the two halves of the molecule is a "gap" in which the binding of oligosaccharides occurs. The walls of the gap are formed mainly by the side chains of non-polar amino acids, which ensure the binding of non-polar molecules of the substrate, and also include the side chains of polar amino acids, which are capable of forming hydrogen bonds with the acylamino and hydroxyl groups of the substrate. The size of the gap allows to accommodate an oligosaccharide molecule containing 6 monosaccharide residues. It is not possible to establish the nature of the binding of the substrate, for example, the hexasaccharide NAG 6 , by the method of X-ray diffraction analysis. At the same time, complexes of the enzyme with the trisaccharide inhibitor NAG 3 are stable and well studied. NAG 3 binds in a gap on the surface of the enzyme, forming hydrogen bonds and van der Waals contacts; at the same time, it fills only half of the gap, in which three more monosaccharide residues can bind. The non-reducing end (sugar A) is at the beginning of the gap, and the reducing end (sugar C) is in its central part; sugar residues A, B and C have a chair conformation. The construction of a model of the enzyme-substrate complex was based on the assumption that the same interactions are realized upon binding of the NAG 6 substrate as upon the binding of NAG 3 . In the enzyme model, three sugar residues (referred to as residues D, E, and F) were placed inside the gap; each subsequent sugar was attached in such a way that its conformation was the same (as far as possible) as that of the first three sugars. As part of the model complex, all sugar residues implement effective non-covalent interactions with side and peptide groups of amino acid residues that form a gap.
When identifying catalytic groups, it was natural to focus on those that are in the enzyme-substrate complex near the cleavable glycosidic bond and can serve as proton donors or acceptors. It turned out that on one side of the split bond, at a distance? 0.3 nm (from the oxygen of the glycosidic bond), the carboxyl group of Glu-35 is located, and on the other (at the same distance) the carboxyl group of Asp-52, their environment is very different. Glu-35 is surrounded by hydrophobic residues; it can be assumed that at the optimum pH of the enzyme, this group is in a non-ionized state. The environment of Asp-52 is pronounced polar; its carboxyl group participates as a hydrogen acceptor in a complex network of hydrogen bonds and probably functions in an ionized state.
The following scheme of the catalytic process during the hydrolysis of the oligosaccharide has been proposed. The non-ionized carboxyl group of Glu-35 acts as a proton donor, supplying it to the glycosidic oxygen atom between the C (1) atom of sugar D and the C (4) atom of sugar E (general acid catalysis stage); this results in the breaking of the glycosidic bond. As a result, the sugar residue D passes into the state of a carbocation with a positively charged carbon atom C (1) and takes on a half-chair conformation. The negative charge of the Asp-52 carboxylate group stabilizes the carbocation. The remainder of NAG 2 (sugars E+F) diffuses out of the active site region. Then a water molecule enters the reaction; its proton passes to Glu-35, and the OH - group to the C (1) atom of the D residue (the stage of basic catalysis). The NAG 4 residue (sugars A + B + C + D) leaves the region of the active center, and the enzyme returns to its original state.
Ribonuclease (RNase) of the bovine pancreas hydrolyzes internucleotide bonds in RNA near the pyrymylin units, which remain esterified at the 3 "position. The enzyme, along with other nucleases, is widely used in the analysis of the structure of RNA.
RNase is formed by one polypeptide chain containing 124 amino acid residues, and its molecular weight is 13,680; There are four disulfide bonds in the molecule. RNase is the first enzyme for which a primary structure has been established.
Based on the results of the study of ribonuclease renaturation, K. Afinsen for the first time clearly formulated the idea that the spatial structure of a protein is determined by its primary structure.
In 1958, F. Richards showed that, under certain conditions, subtilisin cleaves the peptide bond Ala-20 - Ser-21 in RNase. The resulting fragments were called S-peptide (residues 1-20) and S-protein (residues 21-124); due to non-covalent interactions, the fragments form a complex called RNase S. This complex has almost the full catalytic activity of the native enzyme; in isolated form, S-peptide and S-protein are inactive. Further, it was found that a synthetic peptide identical in sequence to the S-peptide fragment containing residues 1 to 13 restores the activity of the S-protein, but a shorter peptide containing residues 1 to 11 does not have this ability. The data obtained allowed us to conclude that the corresponding His-12 or Met-13 residues (or both of these residues) are included in the active site of the enzyme.
When studying the effect of pH on RNase activity, the important role of protein functional groups with pK 5.2 and 6.8 was elucidated; this suggested the participation of histidine residues in the catalytic process.
Upon carboxylation of RNase with iodoacetate at pH 5.5, i.e. under conditions under which the modification of histidine residues predominantly occurs, a complete loss of activity was observed; the modified enzyme contains 1 mol of carboxymethyl groups per 1 mol of protein. As a result, two monocarboxymethylene forms of the enzyme are formed. In one form, His-12 is carboxymethylated, and in the other, His-119. His-119 was predominantly modified.
These data suggested that His-12 and His-119 are in the active site and that modification of one of them prevents modification of the other.
As a result of X-ray diffraction studies, the spatial structure of RNase S and the complex of RNase S with inhibitors was elucidated. The molecule has the shape of a kidney, the active center is localized in the depression where the residues of His-12, His-119 and Lys-41 are located.
Hydrolysis occurs as a result of the conjugated action of His-12 and His-119 residues, which carry out acid-base catalysis. The diagram below shows the stages of the catalytic process:
1. The substrate is in the active site; His-12, His-119 and Lys-41 are located near the negatively charged phosphate.
2. As a result of the action of His-12 as a base that accepts a proton from the 2 "-OH group of ribose, and His-119 as an acid that donates a proton to the oxygen atom of the phosphate, an intermediate complex is formed first, and then a 2", 3 "-cyclic phosphate .
3. In place of the departed product, water enters, donating the proton of His-119, and OH to phosphate, at the same time the proton from His-12 passes to the oxygen atom of ribose, the second product is formed, and the enzyme returns to its original state.
Chymotrypsin is secreted in the form of a proenzyme - chymotrypsinogen by the pancreas of vertebrates; proenzyme activation occurs in the duodenum under the action of trypsin. The physiological function of chymotrypsin is the hydrolysis of proteins and polypeptides. Chymotrypsin attacks mainly peptide bonds formed by carboxyl residues of tyrosine, tryptophan, cenylalanine and methionanine. It also effectively hydrolyses the esters of the corresponding amino acids. The molecular weight of chymotrypsin is 25,000, the molecule contains 241 amino acid residues. Chymotrypsin is formed by three polypeptide chains linked by disulfide bridges.
The functional groups of the active site of chymotrypsin have been identified using irreversible inhibitors. The Ser-195 residue was modified with diisopropyl fluorophosphate and phenylmethylsulfofluoride, and the His-122 residue was modified with N-tosyl-L-phenylalanine-chloromethyl ketone. The two-stage process of chymotrypsin hydrolysis was discovered in the study of the kinetics of hydrolysis of p-nitrophenylacetate.
A characteristic feature of the process under consideration is the formation of a covalent intermediate, an acyl enzyme. The acylated catalytic group was identified as the residue Ser-195. The mechanism of catalysis carried out by the enzyme was proposed even before the establishment of the spatial structure of the protein, but was later refined. In particular, studies using 18 H 2 O made it possible to prove the formation of an acyl enzyme during the hydrolysis of peptides.
A three-dimensional structure with a resolution of 0.2 nm was established by D. Blow's X-ray diffraction analysis. in 1976 The molecule has the shape of an ellipsoid with axes 5.4*4*4 nm. The results of crystallographic studies confirmed the assumption that the Ser-195 and His-57 residues are close. The hydroxyl group of Ser-195 is located at a distance of ~0.3 nm orth of the nitrogen atom of the His-57 imidazole ring. The most interesting fact was that the nitrogen atom in position 1 of the ring is located at a distance of ~0.28 nm from the oxygen atom of the carboxyl group of the Asp-102 side chain and occupies a position favorable for the formation of a hydrogen bond.
It should be noted that chemical studies could not reveal the involvement of Asp-102 in the functioning of the active center, since this residue is embedded deep into the molecule.
It is currently believed that the three residues Asp-102, His-57 and Ser-195 form a charge transfer system that plays a critical role in the catalysis process. The functioning of the system ensures the effective participation of His-57 in catalysis as an acid-base catalyst and increases the reactivity of Ser-195 to the carboxyl carbon of the attacked bond.
The key element of catalysis is the proton transfer from Ser-195 to His-57. At the same time, the oxygen atom of serine attacks the carbonyl carbon atom of the substrate with the formation of first an intermediate tetrahedral compound (1), and then an acyl enzyme (2). The next step is deacylation. The water molecule enters the charge transfer system, and the OH ion simultaneously attacks the carbonyl carbon atom of the acyl group of the acyl enzyme. As in the acylation step, an intermediate tetrahedral compound (4) is formed. His-57 then donates a proton to the oxygen atom of Ser-195, releasing the acyl product; it diffuses into the solution, and the enzyme returns to its original state.
Carboxypeptidase A is secreted as a proenzyme by the pancreas of vertebrates. The formation of the active enzyme occurs in the small intestine with the participation of chymotrypsin. The enzyme sequentially cleaves off C-terminal amino acid residues from the peptide chain, i.e. is an exopeptidase.
Carboxypeptidase A is formed by a single polypeptide chain containing 307 amino acid residues; the molecular weight is 34,470. The amino acid sequence of the protein was established in 1969 by R. Bredshaw.
Elucidation of the mechanism of action of the enzyme was possible only after X-ray diffraction studies. The spatial structure of the enzyme and its complex with the Gly-Tyr dipeptide (substrate model) was established by W. Lipscomb. The enzyme molecule has the shape of an ellipsoid with axes 5.0*4.2*3.8 nm; the active center is located in a depression that passes into a deep non-polar pocket. A zinc ion is localized in the active center zone (its ligands are the side chains of Glu-72, His196, His-69 residues and a water molecule), as well as functional groups involved in substrate binding and catalysis - Arg-145, Glu-270 and Tyr-248.
A comparative analysis of the structures of the enzyme and its complex with Gly-Tyr yielded important information on the structure of the enzyme-substrate complex. In particular, it was found that during the formation of the complex, the hydroxyl group of Tyr-248 moves 1.2 nm relative to its position in the free enzyme (ie, approximately 1/3 of the molecule diameter).
According to the scheme of the catalytic process, the carboxylate group of Glu-270 activates a water molecule located in the reaction sphere, pulling a proton from it; the resulting OH- ion carries out a nucleophilic attack on the carbonyl carbon of the cleavable bond. At the same time, the hydroxyl group of Tyr-248, located near the nitrogen atom of the cleavable peptide bond, donates a proton to it. As a result, the attacked peptide bond is cleaved and the resulting products leave the active site zone. The diagram below illustrates the general basic catalysis.
Aspartate aminotransferase catalyzes the reversible transamination reaction.
The enzymatic transamination reaction was discovered by A.E. Braunstein and M.G. Kritzman in 1937 in the study of an enzyme preparation from the muscle of a pigeon. In subsequent studies, it was shown that transamination reactions are widespread in wildlife and play an important role in the conjugation of nitrogen and energy metabolism.
In 1945, it was found that pyridoxal-5 "-phosphate (PLP) is a coenzyme of aminotransferases. The AAT molecule is a dimer formed by identical subunits. In the cardiac muscle of the investigated vertebrates, there are two isoenzymes - cytoplasmic (cAAT0) and mitochondrial (mAAT) aminotransferases.
The primary structure of cAAT from cardiac muscle was established in 1972. Yu.A. Ovchinnikov and A.E. Brainstein. The polypeptide chain of a protein contains 412 amino acid residues; molecular weight is 46,000.
The general theory of pyridoxal catalysis was developed by A.E. Braunstein and M.M. Shemyakin in 1952-1953, and somewhat later - D.E. Metzler and E.E. Snell. According to this theory, the catalytic action of pyridoxal enzymes is due to the ability of the aldehyde group of pyridoxal phosphate to form aldimines (Schiff bases) when interacting with amines, including amino acids.
In the resulting phosphopyridoxyldeneamino acid, there is a system of conjugated double bonds, along which the displacement of electrons from the 6-carbon atom facilitates the breaking of the bonds formed by this atom.
Modern ideas about the mechanism of enzymatic transamination, developed by A.E. Braunstein and his collaborators are a development of the above theory. In the initial state, the aldehyde group of pyridoxal phosphate forms an aldimine bond with the e-amino group of the Lys-258 residue of the active center (I). Upon binding of the amino acid, a Michaelis complex (II) is formed, followed by an aldimine between pyridoxal phosphate and substrate (III). As a result of subsequent transformations through intermediate stages (IV) and (V), oxo acid (VI) is formed. This completes the first half-reaction of transamination. Repeating these same steps in the "reverse" direction with the new hydroxy acid constitutes the second half-reaction that completes the catalytic transamination cycle.
Myoglobin and hemoglobin
These two proteins are often referred to as respiratory enzymes. Their interaction with the substrate, oxygen, has been elucidated in detail, primarily on the basis of high-resolution X-ray diffraction analysis. The three-dimensional structure of myoglobin was determined by J. Kendrew in 1961, and the three-dimensional structure of hemoglobin - by M. Perutz in 1960.
The myoglobin molecule has a compact shape - 4.5 * 3.5 * 2.5 nm, the polypeptide chain forms 8 helical sections, denoted by letters from A to H. It is arranged in a specialized way around a large flat iron-containing heme ring. Heme is a complex of porphyrin with ferrous iron.
The polar heme propionic acid chains are located on the surface of the molecule, the rest of the heme is embedded in the globule. The connection of heme with the protein is carried out due to the coordination bond between the iron atom and the histidine atom, localized in the F helix; this is the so-called proximal histidine. Another important histidine residue, distal histidine, is localized in the heme pocket in the E helix; it is located on the opposite side of the iron atom at a greater distance than the proximal histidine. The region between the gene iron and the distal histidine in deoxymyoglobin is free, and the lipophilic O 2 molecule can bind to the heme iron, occupying the sixth coordination position. A unique feature of myoglobin, as well as hemoglobin, is their ability to reversibly bind O 2 without oxidizing heme Fe 2+ to Fe 3+ . This is possible because a low permittivity medium is created in the hydrophobic heme pocket from which water is displaced.
When O 2 is bound to an iron atom, the latter moves by about 0.06 nm and ends up in the plane of the porphyrin ring, i.e. in an energetically more favorable position. It is believed that this movement is due to the fact that the Fe 2+ ion in deoxymyoglobin is in a high-spin state and its radius is too large to fit in the plane of the heme porphyrin ring. When O 2 is bound, the Fe 2+ ion passes into a low-pin state and its radius decreases; now the Fe 2+ ion can move into the plane of the porphyrin ring.
Hemoglobin is the main component of red blood cells that delivers oxygen from the lungs to the tissues, and carbon dioxide from the tissues to the lungs. Hemoglobins of different types differ in the form of crystals, solubility, affinity for oxygen. This is due to differences in the amino acid sequence of proteins; the heme component is the same in hemoglobins of all vertebrate species and some invertebrates.
Human hemoglobin is a tetramer consisting of four subunits, two b-subunits and two b-subunits, each containing 141 and 146 amino acid residues, respectively. There is significant homology between the primary structures of the β and β subunits, and the conformation of their polypeptide chains is also similar.
The hemoglobin molecule has a spherical shape with a diameter of 5.5 nm. The four subunits are packed in a tetrahedral shape.
X-ray diffraction data showed that oxygenation of hemoglobin is accompanied by a number of changes. At low resolution, it was found that in this case the structure becomes more compact (the Fe atoms of the β-chains approach each other by about 0.6-0.7 nm), the subunits rotate relative to each other and the second-order axis by 10-15 o. The results of the study at high resolution indicate that especially significant changes occur in the region of 6v contacts.
To date, on the basis of X-ray diffraction studies and a number of other methodological approaches, significant progress has been made in elucidating the mechanism of action of enzymes with desired properties based on achievements in the field of genetic engineering. This opens up wide opportunities for testing the validity of modern ideas about the mechanism of enzyme action and creating a fundamental theory of enzymatic catal.