Catalysis is the process of accelerating a chemical reaction under the influence of catalysts that actively participate in it, but at the end of the reaction remain chemically unchanged. The catalyst accelerates the establishment of chemical equilibrium between the starting materials and reaction products. The energy required to start a chemical reaction is called activation energy. It is necessary so that the molecules participating in the reaction can enter a reactive (active) state. The mechanism of action of the enzyme is aimed at reducing the activation energy. This is achieved by dividing the reaction into separate steps or stages through the participation of the enzyme itself. Each new stage has a lower activation energy. The division of the reaction into stages becomes possible due to the formation of a complex of the enzyme with the starting substances, the so-called substrates ( S). Such a complex is called an enzyme-substrate complex ( ES). This complex is then cleaved to form the reaction product (P) and the unchanged enzyme ( E).
E + SESE + P
Thus, an enzyme is a biocatalyst that, by forming an enzyme-substrate complex, breaks the reaction into separate stages with a lower activation energy and thereby sharply increases the reaction rate.
4. Properties of enzymes.
All enzymes are of protein nature.
Enzymes have high molecular weight.
They are highly soluble in water and, when dissolved, form colloidal solutions.
All enzymes are thermolabile, i.e. optimum action 35 – 45 o C
According to their chemical properties, they are amphoteric electrolytes.
Enzymes are highly specific with respect to substrates.
Enzymes require a strictly defined pH value for their action (pepsin 1.5 - 2.5).
Enzymes have high catalytic activity (accelerate the reaction rate by 10 6 – 10 11 times).
All enzymes are capable of denaturation when exposed to strong acids, alkalis, alcohols, and heavy metal salts.
Specificity of enzyme action:
Based on the specificity of their action, enzymes are divided into two groups: those with absolute specificity and those with relative specificity.
Relative specificity observed when an enzyme catalyzes one type of reaction with more than one structure-like substrate. For example, pepsin breaks down all proteins of animal origin. Such enzymes act on a specific type of chemical bond, in this case a peptide bond. The action of these enzymes extends to a large number of substrates, which allows the body to get by with a small number of digestive enzymes.
Absolute specificity manifests itself when the enzyme acts on only one single substance and catalyzes only a certain transformation of this substance. For example, sucrase only breaks down sucrose.
Reversibility of action:
Some enzymes can catalyze both forward and reverse reactions. For example, lactate dehydrogenase, an enzyme that catalyzes the oxidation of lactate to pyruvate and the reduction of pyruvate to lactate.
Denaturation, causes and symptoms, use in medicine.
Proteins are sensitive to external influences. Violation of the spatial structure of proteins is called denaturation. In this case, the protein loses all its biological and physicochemical properties. Denaturation is accompanied by the rupture of bonds that stabilize the “native” structure of the protein. As noted above, weak interaction plays the main role in stabilizing the structure of proteins, so denaturation can be caused by various factors: heating, irradiation, mechanical shaking, cooling, chemical exposure. During denaturation, as a rule, the solubility of proteins is also impaired, since a violation of the structure leads to the appearance on the surface of a large number of hydrophobic groups, usually hidden in the center of the protein molecule.
The primary structure of a protein does not change during denaturation, which made it possible to show the possibility of restoring the functions and structure of a denatured protein, although in most cases denaturation is an irreversible process. In laboratory practice, denaturation is used to deproteinize biological fluids. Factors that cause denaturation are called denaturing agents. These include:
1. Heating and high-energy irradiation (ultraviolet, x-ray, neutron, etc.). It is based on the excitation of atomic vibrations, accompanied by the breaking of bonds.
2. Action of acids and alkalis; change the dissociation of groups, reduce the number of ionic bonds.
3. Heavy metal ions. They form complex compounds with protein groups, which is accompanied by the rupture of weak interactions.
4. Reducing agents - cause the rupture of disulfide bridges.
5. Urea, guanidinium chloride - form new hydrogen bonds and break old ones. The phenomenon of denaturation can also be used for qualitative analysis of the presence of proteins in solutions. To do this, use a sample with boiling of the liquid being tested after it has been acidified. The resulting turbidity is due to protein denaturation. Precipitation with organic acids is also often used: sulfosalicylic or trichloroacetic.
A brief history of enzymology.
The awarding of the Nobel Prize to J. Sumner, J. Northrop and Stanley in 1946 marked the end of a long period of development of enzymology - the science of enzymes. The beginning of this science goes back to the dawn of the history of the development of mankind, which uses a number of technological enzymatic processes in its life: bread baking, winemaking, processing of animal skins, etc. The need to improve these processes became the impetus for their in-depth study. The first scientific descriptions of enzymatic processes include the description of digestion in animals. When setting up his experiments, René Antoine Reaumur (1683-1757) proceeded from the assumption made by Faulkner that birds of prey regurgitate undigested food remains. Reaumur constructed a small wire capsule into which he placed a piece of meat and gave it to a buzzard to peck at. After 24 hours, the bird spat out this capsule. A softened piece of food remained in it, which, however, did not spoil. “This process can only be the result of the action of some kind of solvent,” concluded Reaumur. Lazzaro Spallanzani (1729-1799), professor of natural history at the University of Padua, reported similar experiments. However, he did not consider digestion as a fermentation process for the simple reason that gas bubbles were not formed.
Later, the fermentation process was studied in more detail by one of the founders of modern chemistry, Antoine Laurent Lavoisier (1743-1794). While studying the alcoholic fermentation that occurs during the production of wine, he discovered that glucose is converted into alcohol and carbon dioxide,
By the beginning of the 19th century. The prevailing general view was that fermentation was a chemical change brought about by certain specialized forms of organic material, namely "enzymes". In 1814, the Russian scientist (German by birth) academician of the St. Petersburg Academy of Sciences Konstantin Gottlieb Sigismund Kirchhoff (1764-1833) showed that the formation of sugar from starch in sprouted cereal grains is due to a chemical process and not the appearance of sprouts. In 1810, Yu. Gay-Lussac isolated the main end products of yeast activity - alcohol and carbon dioxide. J. Berzelius, one of the founders of the theory of chemical catalysis and the author of the term “catalysis” itself in 1835, confirms these data, noting that diastase (extract from malt) catalyzes the hydrolysis of starch more effectively than mineral sulfuric acid. An important role in the development of enzymology was played by the dispute between Yu Liebig and the famous microbiologist L. Pasteur, who believed that fermentation processes can only occur in a whole living cell. J. Liebig, on the contrary, believed that biological processes are caused by the action of chemicals, which were later called enzymes. The term enzyme (Greek en - in, zyme - yeast) was proposed in 1878 by Friedrich Wilhelm Kühne to emphasize that the process occurs in the yeast, as opposed to the yeast itself, which catalyzes the fermentation process. However, in 1897, E. Buchner obtained a cell-free yeast extract capable of producing ethanol and confirmed Liebig's opinion.
Attempts to explain one of the important properties of enzymes, specificity, led in 1894 the German chemist and biochemist E. Fischer to propose a model of interaction between the enzyme and the substrate, called the “key-lock” - geometric complementarity of the shapes of the substrate (key) and the enzyme (lock). In 1926, J. Sumner, after almost 9 years of research, proved the protein nature of the urease enzyme. In the same years, J. Northrop and M. Kunitz pointed out a direct correlation between the activity of crystalline pepsin, trypsin and the amount of protein in the studied samples, thereby providing significant evidence of the protein nature of the enzymes, although the final evidence was obtained after determining the primary structure and artificial synthesis of a number of enzymes. Basic ideas about enzymes were obtained already in the second half of the twentieth century. In 1963, the amino acid sequence of RNase from the pancreas was studied. In 1965, the spatial structure of lysozyme was shown. Over the following years, thousands of enzymes were purified and a lot of new data was obtained on the mechanisms of action of enzymes, their spatial structure, and the regulation of reactions catalyzed by enzymes. Catalytic activity was discovered in RNA (ribozymes). Antibodies with enzymatic activity—abzymes—were obtained. This chapter briefly introduces modern ideas about the structure, mechanism of action and medical aspects of enzymology.
Features of enzymatic catalysis.
1. Protein nature of the catalyst
2. Exceptionally high efficiency. The efficiency of biological catalysis exceeds the efficiency of inorganic catalysis by 10 9 - 10 12
3. Exceptionally high specificity:
a) absolute, when the enzyme works only with its substrate (fumarase with trans-isomers of fumaric acid and not with cis-isomers);
b) group - specific for a narrow group of related substrates (gastrointestinal enzymes).
4. Works under mild conditions (t=37, pH 7.0, certain osmolarity and salt composition).
5. Multi-level regulation: regulation of activity at the level of environmental conditions, at the metabolon level, at the genetic level, tissue, cellular, with the help of hormones and mediators, as well as with the help of substrates and products of the reaction that they catalyze.
6. Cooperativeness: enzymes are capable of organizing associations - the product of the 1st enzyme is a substrate for the 2nd; the product of the 2nd is a substrate for the 3rd, etc.
In addition, enzymes are adaptable, i.e. they can change their activity and form new associations.
7. Capable of catalyzing both forward and reverse reactions. The direction of the reaction for many enzymes is determined by the ratio of the acting masses.
8. Catalysis is strictly scheduled, that is, it occurs in stages.
Specificity of enzyme action.
The high specificity of enzymes is due to the conformational and electrostatic complementarity between the molecules of the substrate and the enzyme and the unique structure of the active center of the enzyme, which ensures “recognition”, high affinity and selectivity for the occurrence of one particular reaction.
Depending on the mechanism of action, enzymes with relative or group specificity and with absolute specificity are distinguished.
For the action of some hydrolytic enzymes, the type of chemical bond in the substrate molecule is of greatest importance. For example, pepsin breaks down proteins of animal and plant origin, although they may differ in chemical structure, composition, and physiological properties. However, pepsin does not break down carbohydrates and fats. This is explained by the fact that the site of action of pepsin is the peptide bond. For the action of lipase, such a site is the ester bond of fats.
That is, these enzymes have relative specificity.
Absolute specificity of action is called the ability of an enzyme to catalyze the transformation of only a single substrate and any changes in the structure of the substrate make it inaccessible to the action of the enzyme. For example: arginase, which breaks down arginine; urease, which catalyzes the breakdown of urea.
There is evidence for the existence of stereochemical specificity due to the existence of optically isomeric L- and D-forms or geometric (cis- and trans-) isomers
Thus, oxidases L and D a/k are known.
If any compound exists in the form of cis- and trans-isomers, then for each of these forms there is its own enzyme. For example, fumarase catalyzes the conversion of only fumaric acid (trans), but does not act on the cis isomer, maleic acid.
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 converting 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 acidic 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). This property of active site functional groups makes enzymes unique biological catalysts, in contrast to non-biological catalysts that can exhibit either acidic or basic properties. 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.
25. Complementarity refers to the spatial and chemical correspondence of interacting molecules. The ligand must have the ability to enter and spatially coincide with the conformation of the active site. This coincidence may not be complete, but due to the conformational lability of the protein, the active center is capable of small changes and is “adjusted” to the ligand. In addition, between the functional groups of the ligand and the amino acid radicals forming the active center, bonds must arise that hold the ligand in the active center. The bonds between the ligand and the active center of the protein can be either non-covalent (ionic, hydrogen, hydrophobic) or covalent.
The fact that enzymes have high specificity allowed us to put forward a hypothesis in 1890, according to which the active center of the enzyme is complementary to the substrate, i.e. corresponds to it like a “key to a 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, strictly determined structure.
The substrate, interacting with the active center of the enzyme, causes a change in its conformation, leading to the formation of an enzyme-substrate complex, favorable for chemical modifications of the substrate. At the same time, the substrate molecule also changes its conformation, which ensures higher efficiency of the enzymatic reaction. This “induced correspondence hypothesis” was subsequently confirmed experimentally.
26. Enzymes that catalyze the same chemical reaction, but differ in the primary protein structure, are called isoenzymes, or isoenzymes. They catalyze the same type of reaction with a fundamentally identical mechanism, but differ from each other in kinetic parameters, activation conditions, and features of the connection between the apoenzyme and the coenzyme. The nature of the appearance of isoenzymes is varied, but most often due to differences in the structure of the genes encoding these isoenzymes. Consequently, isoenzymes differ in the primary structure of the protein molecule and, accordingly, in physicochemical properties. Methods for determining isoenzymes are based on differences in physicochemical properties. In their structure, isoenzymes are mainly oligomeric proteins. Enzyme lactate dehydrogenase(LDH) catalyzes the reversible oxidation reaction of lactate (lactic acid) to pyruvate (pyruvic acid).
It consists of 4 subunits of 2 types: M and H. The combination of these subunits underlies the formation of 5 isoforms of lactate dehydrogenase. LDH 1 and LDH 2 are most active in the heart muscle and kidneys, LDH4 and LDH5 - in skeletal muscles and liver. Other tissues contain various forms of this enzyme. LDH isoforms differ in electrophoretic mobility, which makes it possible to determine the tissue identity of LDH isoforms.
Creatine kinase (CK) catalyzes the formation of creatine phosphate:
The KK molecule is a dimer consisting of two types of subunits: M and B. From these subunits, 3 isoenzymes are formed - BB, MB, MM. The BB isoenzyme is found primarily in the brain, MM in skeletal muscles, and MB in cardiac muscle. KK isoforms have different electrophoretic mobilities. CK activity should normally not exceed 90 IU/l. Determination of CK activity in blood plasma has diagnostic value in case of myocardial infarction (there is an increase in the level of the MB isoform). The amount of the MM isoform may increase during trauma and damage to skeletal muscles. The BB isoform cannot penetrate the blood-brain barrier, therefore it is practically undetectable in the blood even during strokes and has no diagnostic value.
27. ENZYMATIVE CATALYSIS (biocatalysis), acceleration of biochemical. r-tions with the participation of protein macromolecules called enzymes(enzymes). F.k. - variety catalysis.
Michaelis-Menten equation: - the basic equation of enzyme kinetics, describes the dependence of the rate of reaction catalyzed by an enzyme on the concentration of the substrate and enzyme. The simplest kinetic scheme for which the Michaelis equation is valid:
The equation looks like:
,
Where: - maximum reaction rate, equal to ; - Michaelis constant, equal to the substrate concentration at which the reaction rate is half the maximum; - substrate concentration.
Michaelis constant: Relationship between rate constants
is also a constant ( K m).
28. "inhibition of enzymatic activity" - a decrease in catalytic activity in the presence of certain substances - inhibitors. Inhibitors should include substances that cause a decrease in enzyme activity. Reversible inhibitors bind to the enzyme with weak non-covalent bonds and, under certain conditions, are easily separated from the enzyme. There are reversible inhibitors competitive and non-competitive. Towards competitive inhibition include a reversible decrease in the rate of an enzymatic reaction caused by an inhibitor that binds to the active site of the enzyme and prevents the formation of an enzyme-substrate complex. This type of inhibition is observed when the inhibitor is a structural analogue of the substrate, resulting in competition between substrate and inhibitor molecules for a place in the active center of the enzyme. Non-competitive called inhibition of an enzymatic reaction in which the inhibitor interacts with the enzyme at a site other than the active site. Noncompetitive inhibitors are not structural analogues of the substrate. Irreversible inhibition observed in the case of the formation of covalent stable bonds between the inhibitor molecule and the enzyme. Most often, the active center of the enzyme is modified. As a result, the enzyme cannot perform a catalytic function. Irreversible inhibitors include heavy metal ions, such as mercury (Hg 2+), silver (Ag +) and arsenic (As 3+). Substances that block certain groups of the active center of enzymes - specific And. Diisopropyl fluorophosphate (DFP). Iodine acetate and p-chloromercuribenzoate readily react with the SH groups of cysteine residues in proteins. These inhibitors are classified as nonspecific. At non-competitive In inhibition, the inhibitor binds only to the enzyme-substrate complex and not to the free enzyme.
Size K I= [E]. [I]/, which is the dissociation constant of the enzyme-inhibitor complex, is called the inhibition constant.
Quaternary ammonium bases inhibit acetylcholinesterase, which catalyzes the hydrolysis of acetylcholine into choline and acetic acid.
Substances called antimetabolites. These compounds, being structural analogs of natural substrates, cause competitive inhibition of enzymes, on the one hand, and, on the other, can be used by the same enzymes as pseudosubstrates. Sulfonamide drugs (analogues of para-aminobenzoic acid) used to treat infectious diseases.
An example of a drug whose action is based on irreversible enzyme inhibition is the drug aspirin.
Inhibition of the enzyme cyclooxygenase, which catalyzes the formation of prostaglandins from arachidonic acid.
29. Regulation of the rate of enzymatic reactions is carried out at 3 independent levels:
1. changing the number of enzyme molecules;
- availability of substrate and coenzyme molecules;
- a change in the catalytic activity of the enzyme molecule.
1. The number of enzyme molecules in a cell is determined by the ratio of 2 processes - synthesis and breakdown of the enzyme protein molecule.
2. The higher the concentration of the initial substrate, the higher the speed of the metabolic pathway. Another parameter limiting the course of the metabolic pathway is the presence regenerated coenzymes. The most important role in changing the speed of metabolic pathways is the regulation of the catalytic activity of one or more key enzymes of a given metabolic pathway. This is a highly effective and fast way to regulate metabolism. The main ways to regulate enzyme activity are: allosteric regulation; regulation by protein-protein interactions; regulation by phosphorylation/dephosphorylation of the enzyme molecule; regulation by partial (limited) proteolysis.
Increasing the temperature to certain limits affects the rate of enzymatic
reaction, similar to the effect of temperature on any chemical reaction. As the temperature increases, the movement of molecules accelerates, which leads to an increase in the likelihood of interaction between reactants. In addition, temperature can increase the energy of reacting molecules, which also speeds up the reaction. However, the rate of a chemical reaction catalyzed by enzymes has its own temperature optimum, exceeding which is accompanied by a decrease in enzymatic activity
For most human enzymes, the optimal temperature is 37-38 °C.
The activity of enzymes depends on the pH of the solution in which the enzymatic reaction occurs. For each enzyme there is a pH value at which its maximum activity is observed. Deviation from the optimal pH value leads to a decrease in enzymatic activity.
The effect of pH on enzyme activity is associated with the ionization of functional groups of amino acid residues of a given protein, which ensure the optimal conformation of the active center of the enzyme. When pH changes from optimal values, the ionization of the functional groups of the protein molecule changes. Most of the enzymes in the human body have an optimum pH close to neutral, coinciding with the physiological pH value
30. Allosteric enzymes are enzymes whose activity is regulated not only by the number of substrate molecules, but also by other substances called effectors. The effectors involved in allosteric regulation are cellular metabolites often of the very pathway they regulate.
Allosteric enzymes play an important role in metabolism, as they react extremely quickly to the slightest changes in the internal state of the cell. They are of great importance in the following situations: during anabolic processes, during catabolic processes, for the coordination of anabolic and catabolic pathways. ATP and ADP are allosteric effectors that act as antagonists; to coordinate parallel and interconnected metabolic pathways (for example, the synthesis of purine and pyrimidine nucleotides used for the synthesis of nucleic acids).
An effector that causes a decrease (inhibition) of enzyme activity is called negative effector or inhibitor. An effector that causes an increase (activation) of enzyme activity is called positive effector or activator. Various metabolites often serve as allosteric effectors.
Features of the structure and functioning of allosteric enzymes: usually these are oligomeric proteins consisting of several protomers or having a domain structure; they have an allosteric center, spatially distant from the catalytic active center; effectors attach to the enzyme non-covalently in allosteric (regulatory) centers; allosteric centers, like catalytic ones, can exhibit different specificity in relation to ligands: it can be absolute and group. the protomer on which the allosteric center is located is a regulatory protomer. Allosteric enzymes have the property of cooperativity; allosteric enzymes catalyze key reactions in this metabolic pathway.
the final product can act as an allosteric inhibitor of the enzyme that most often catalyzes the initial stage of this metabolic pathway:
In central metabolic pathways, precursors can be activators of key enzymes in the metabolic pathway.
Any catalytic reaction involves a change in the rates of both forward and reverse reactions due to a decrease in its energy. If a chemical reaction proceeds with the release of energy, then it must begin spontaneously. However, this does not happen because the components of the reaction must be transferred to the activated (transition) state. The energy required to convert reacting molecules into an activated state is called activation energy.
Transition state characterized by the continuous formation and breaking of chemical bonds, and thermodynamic equilibrium exists between the transition and ground states. The rate of the forward reaction depends on the temperature and the difference in free energy values for the substrate in the transition and ground states. This difference is called free energy of reaction.
Achieving the transition state of the substrate is possible in two ways:
- due to the transfer of excess energy to reacting molecules (for example, due to an increase in temperature),
- by reducing the activation energy of the corresponding chemical reaction.
Ground and transition states of reacting substances.
Eo, Ek - reaction activation energy without and in the presence of a catalyst; DG-
difference in free energy of reaction.
Enzymes “help” substrates adopt a transition state due to the binding energy during formation enzyme-substrate complex. The decrease in activation energy during enzymatic catalysis is due to an increase in the number of stages of the chemical process. The induction of a number of intermediate reactions leads to the fact that the initial activation barrier is split into several lower barriers, which the reacting molecules can overcome much faster than the main one.
The mechanism of the enzymatic reaction can be represented as follows:
- connection of enzyme (E) and substrate (S) with the formation of an unstable enzyme-substrate complex (ES): E + S → E-S;
- formation of an activated transition state: E-S → (ES)*;
- release of reaction products (P) and regeneration of the enzyme (E): (ES)* → P + E.
To explain the high efficiency of enzyme action, several theories of the mechanism of enzymatic catalysis have been proposed. The earliest is theory of E. Fisher (the theory of “template” or “rigid matrix"). According to this theory, the enzyme is a rigid structure, the active center of which is a “cast” of the substrate. If the substrate approaches the active site of the enzyme like a “key to a lock,” a chemical reaction will occur. This theory well explains two types of substrate specificity of enzymes - absolute and stereospecificity, but turns out to be untenable in explaining the group (relative) specificity of enzymes.
The "rack" theory based on the ideas of G. K. Euler, who studied the action of hydrolytic enzymes. According to this theory, the enzyme binds to the substrate molecule at two points, and the chemical bond is stretched, the electron density is redistributed, and the chemical bond is broken, accompanied by the addition of water. Before joining the enzyme, the substrate has a “relaxed” configuration. After binding to the active center, the substrate molecule undergoes stretching and deformation (it is located in the active center as if on a rack). The longer the chemical bonds in the substrate, the easier they are to break and the lower the activation energy of the chemical reaction.
Recently it has become widespread theory of “induced correspondence” by D. Koshland, which allows for high conformational lability of the enzyme molecule, flexibility and mobility of the active center. The substrate induces conformational changes in the enzyme molecule in such a way that the active center takes on the spatial orientation necessary for binding the substrate, i.e., the substrate approaches the active center like a “hand to a glove.”
According to the theory of induced correspondence, the mechanism of interaction between enzyme and substrate is as follows:
- The enzyme, based on the principle of complementarity, recognizes and “catches” the substrate molecule. In this process, the protein molecule is aided by the thermal movement of its atoms;
- amino acid residues of the active center are shifted and adjusted in relation to the substrate;
- chemical groups are covalently added to the active site - covalent catalysis.
The sequence of events in enzymatic catalysis can be described by the following diagram. First, a substrate-enzyme complex is formed. In this case, a change in the conformations of the enzyme molecule and the substrate molecule occurs, the latter is fixed in the active center in a tense configuration. This is how the activated complex is formed, or transition state, is a high-energy intermediate structure that is energetically less stable than the parent compounds and products. The most important contribution to the overall catalytic effect is made by the process of stabilization of the transition state - the interaction between amino acid residues of the protein and the substrate, which is in a tense configuration. The difference between the free energy values for the initial reactants and the transition state corresponds to the free energy of activation (ΔG #). The reaction rate depends on the value (ΔG #): the smaller it is, the greater the reaction rate, and vice versa. Essentially, the DG represents an “energy barrier” that must be overcome for a reaction to occur. Stabilizing the transition state lowers this “barrier” or activation energy. At the next stage, the chemical reaction itself occurs, after which the resulting products are released from the enzyme-product complex.
There are several reasons for the high catalytic activity of enzymes, which reduce the energy barrier to the reaction.
1. An enzyme can bind molecules of reacting substrates in such a way that their reactive groups will be located close to each other and from the catalytic groups of the enzyme (effect rapprochement).
2. With the formation of a substrate-enzyme complex, fixation of the substrate and its optimal orientation for breaking and formation of chemical bonds are achieved (effect orientation).
3. Binding of the substrate leads to the removal of its hydration shell (exists on substances dissolved in water).
4. Effect of induced correspondence between substrate and enzyme.
5. Stabilization of the transition state.
6. Certain groups in the enzyme molecule can provide acid-base catalysis(transfer of protons in the substrate) and nucleophilic catalysis(formation of covalent bonds with the substrate, which leads to the formation of structures that are more reactive than the substrate).
One example of acid-base catalysis is the hydrolysis of glycosidic bonds in the murein molecule by lysozyme. Lysozyme is an enzyme present in the cells of various animals and plants: in tear fluid, saliva, chicken protein, milk. Lysozyme from chicken eggs has a molecular weight of 14,600 Da, consists of one polypeptide chain (129 amino acid residues) and has 4 disulfide bridges, which ensures high stability of the enzyme. X-ray structural analysis of the lysozyme molecule showed that it consists of two domains forming a “gap” in which the active center is located. Along this “gap” the hexosaccharide binds, and the enzyme has its own site for binding each of the six sugar rings of murein (A, B, C, D, E and F) (Fig. 6.4).
The murein molecule is held in the active site of lysozyme mainly due to hydrogen bonds and hydrophobic interactions. In close proximity to the site of hydrolysis of the glycosidic bond, there are 2 amino acid residues of the active center: glutamic acid, occupying the 35th position in the polypeptide, and aspartic acid, the 52nd position in the polypeptide (Fig. 6.5).
The side chains of these residues are located on opposite surfaces of the “cleft” in close proximity to the attacked glycosidic bond—at a distance of approximately 0.3 nm. The glutamate residue is in a non-polar environment and is not ionized, and the aspartate residue is in a polar environment; its carboxyl group is deprotonated and participates as a hydrogen acceptor in a complex network of hydrogen bonds.
The hydrolysis process is carried out as follows. The protonated carboxyl group of the Glu-35 residue provides its proton to the glycosidic oxygen atom, which leads to the rupture of the bond between this oxygen atom and the C 1 atom of the sugar ring located in site D (stage of general acid catalysis). As a result, a product is formed that includes the sugar rings located in regions E and F, which can be released from the complex with the enzyme. The conformation of the sugar ring located in region D is distorted, taking on the conformation half-chairs, in which five of the six atoms forming the sugar ring lie practically in the same plane. This structure corresponds to the transition state conformation. In this case, the C 1 atom turns out to be positively charged and the intermediate product is called a carbonium ion (carbocation). The free energy of the transition state decreases due to the stabilization of the carbonium ion by the deprotonated carboxyl group of the Asp-52 residue (Fig. 6.5).
At the next stage, a water molecule enters the reaction and replaces the disaccharide residue diffusing from the region of the active center. The proton of the water molecule goes to Glu-35, and the hydroxyl ion (OH -) to the C 1 atom of the carbonium ion (stage of general basic catalysis). As a result, the second fragment of the cleaved polysaccharide becomes a reaction product (chair conformation) and leaves the active center region, and the enzyme returns to its original state and is ready to carry out the next disaccharide cleavage reaction (Fig. 6.5).
Properties of enzymes
When characterizing the properties of enzymes, we first use the concept of “activity.” Enzyme activity is understood as the amount of enzyme that catalyzes the conversion of a certain amount of substrate per unit of time. To express the activity of enzyme preparations, two alternative units are used: international (E) and “catal” (kat). The international unit of enzyme activity is taken to be the amount of enzyme that catalyzes the conversion of 1 µmol of substrate into a product in 1 minute under standard conditions (usually optimal). One katal denotes the amount of enzyme that catalyzes the conversion of 1 mole of substrate in 1 s. 1 cat=6*10 7 E.
Often enzyme preparations are characterized by specific activity, which reflects the degree of purification of the enzyme. Specific activity is the number of units of enzyme activity per 1 mg of protein.
The activity of enzymes depends to a very large extent on external conditions, among which the temperature and pH of the environment are of paramount importance. An increase in temperature in the range of 0-50° C usually leads to a smooth increase in enzymatic activity, which is associated with the acceleration of the formation of the substrate-enzyme complex and all subsequent catalytic events. However, a further increase in temperature is usually accompanied by an increase in the amount of inactivated enzyme due to denaturation of its protein part, which is expressed in a decrease in activity. Each enzyme is characterized temperature optimum- the temperature value at which its greatest activity is recorded. More often, for enzymes of plant origin, the temperature optimum lies within 50-60 ° C, and for animal enzymes - between 40 and 50 ° C. Enzymes of thermophilic bacteria are characterized by a very high temperature optimum.
The dependence of enzyme activity on pH values of the environment is also complex. Each enzyme is characterized optimum pH environment in which it exhibits maximum activity. As you move away from this optimum in one direction or the other, enzymatic activity decreases. This is explained by a change in the state of the active center of the enzyme (a decrease or increase in the ionization of functional groups), as well as the tertiary structure of the entire protein molecule, which depends on the ratio of cationic and anionic centers in it. Most enzymes have a pH optimum in the neutral range. However, there are enzymes that exhibit maximum activity at pH 1.5 (pepsin) or 9.5 (arginase).
Enzyme activity is subject to significant fluctuations depending on exposure inhibitors(substances that reduce activity) and activators(substances that increase activity). The role of inhibitors and activators can be played by metal cations, some anions, carriers of phosphate groups, reducing equivalents, specific proteins, intermediate and final products of metabolism, etc. These substances can enter the cell from the outside or be produced within it. In the latter case, they talk about the regulation of enzyme activity - an integral link in the overall regulation of metabolism.
Substances that affect enzyme activity can bind to the active and allosteric centers of the enzyme, as well as outside these centers. Particular examples of such phenomena will be discussed in Chapters 7-19. To generalize some patterns of inhibition of enzyme activity, it should be noted that these phenomena in most cases come down to two types - reversible and irreversible. During reversible inhibition no changes are made to the enzyme molecule after its dissociation with the inhibitor. An example is the action substrate analogues, which can bind to the active site of the enzyme, preventing the enzyme from interacting with the true substrate. However, an increase in the substrate concentration leads to the “displacement” of the inhibitor from the active site, and the rate of the catalyzed reaction is restored ( competitive inhibition). Another case of reversible inhibition is the binding of the inhibitor to a prosthetic group of the enzyme, or apoenzyme, outside the active center. For example, the interaction of enzymes with heavy metal ions that attach to the sulfhydryl groups of amino acid residues of the enzyme, protein-protein interactions or covalent modification of the enzyme. This inhibition of activity is called non-competitive.
Irreversible inhibition in most cases it is based on linking the so-called “ suicidal substrates» with active sites of enzymes. In this case, covalent bonds are formed between the substrate and the enzyme, which are broken down very slowly and the enzyme is not able to perform its function for a long time. An example of a “suicidal substrate” is the antibiotic penicillin (Chapter 18, Fig. 18.1).
Because enzymes are characterized by specificity of action, they are classified according to the type of reaction they catalyze. According to the currently accepted classification, enzymes are grouped into 6 classes:
1. Oxidoreductases (redox reactions).
2. Transferases (reactions of transfer of functional groups between substrates).
3. Hydrolases (hydrolysis reactions, the acceptor of the transferred group is a water molecule).
4. Lyases (reactions of elimination of groups in a non-hydrolytic way).
5. Isomerases (isomerization reactions).
6. Ligases, or synthetases (synthesis reactions due to the energy of cleavage of nucleoside triphosphates, most often ATP).
The number of the corresponding enzyme class is fixed in its code numbering (cipher). The enzyme code consists of four numbers separated by dots, indicating the enzyme class, subclass, subsubclass and serial number in the subclass.