Exponential reliability law in the kinetics of enzyme inactivation. General properties of enzymes The influence of pH on the activity of salivary amylase
Relaxation methods are based on the principle that with a rapid external influence on a system (change in temperature, pressure, etc.), the time that the system needs to achieve a new equilibrium (or stationary state) depends on the rate of the chemical reaction (and sometimes on the rate of diffusion of reagents .
Let us consider the simplest reaction of complexation of the active center of an enzyme with a ligand
At the beginning, the system is in equilibrium, which is characterized by the equilibrium constant K 0 =K(T 0) and, accordingly, equilibrium concentrations 
,
,
. Let us assume that the temperature in the system changes sharply T->T 0 +T. This leads to a change in the equilibrium constant K->K 0 +K, which is determined by the relation
(2.50)
where H– standard enthalpy change. After this, the system transitions to a new equilibrium state:
(2.51)
(2.52)
Equation (2.51) is nonlinear. Let us assume that the deviation from equilibrium is small and then
and equation (2.51) is transformed into a linear differential equation:
The solution to this differential equation is:
Magnitude
(2.54)
called relaxation time.
2.5. The influence of temperature and pH on the rate of enzymatic reactions
The influence of these factors on the rate of an elementary chemical reaction was discussed in Chapter 1. The peculiarity is that enzymatic reactions are complex multi-stage reactions (consisting of many elementary reactions). In addition, the state of enzyme molecules in solution is characterized by a set of conformers that reversibly transform into each other. Conformational transitions of the molecule are determined to a large extent by the temperature and pH of the solution.
2.6. Inhibition of enzymatic reactions
Substances that inhibit the catalytic activity of enzymes are called inhibitors . There are two main classes of inhibitors - reversible
(2.55)
(pesticides, sarin, soman, aspirin, etc.)
And irreversible (inactivators )
(2.55)
(carbon monoxide, cyanide ion, analgin, etc.)
2.7. Enzyme inactivation
Biopolymer molecules (enzymes) are thermodynamically unstable and, as a rule, change their structure and properties over time. In most cases, the inactivation process can be described as a transition between two states of the enzyme being active. E a and inactive E i :
(2.56) The kinetics of the process is described by the corresponding differential equation
(2.57)
and is characterized by a time constant
(2.58)
The process of enzyme inactivation can have a different physicochemical nature. The most common is thermal denaturation, which is a significant restructuring of the macromolecule, a change in the tertiary and partially secondary structure.
For inactivation purposes, cavitation ultrasound, radioactive radiation, etc. can be used.
A change in pH can also lead to denaturation of the enzyme. At each pH value, the protein is characterized by a corresponding charge distribution (ionogenic groups). At very low or very high pH, the charge distribution can significantly polarize the molecule, lead to the appearance of isomers and irreversibly conform it with the destruction of the structure of the active center. For example:
Enzyme denaturation is caused by denaturing agents, destroying the secondary structure of protein (for example, urea), as well as oxidative processes involving oxygen.
When studying such processes, important information is obtained by relaxation methods. As a rule, conformational changes are accompanied by changes in the environment of aromatic amino acids - tyrosine and tryptophan (radiation absorption band at 290 nm). This manifests itself in changes in absorption and fluorescence spectra.
Reversible conformational changes usually occur over a time of 0.1-100 ms, and irreversible ones - 1-1000 minutes.
Example 1. The simplest kinetic scheme of inactivation with conformer equilibrium:
(2.59)
The kinetics of the process is described by one characteristic time
(2.60)
Example 2. Both conformers are subject to inactivation:
(2.61)
(2.62)
Example 3. A more general case for a system involving n conformers:
Enzymes often form dimers in solution, and are more stable in the dimeric form. Then it is observed dissociative inactivation mechanism :
(2.65)
The following scheme reflects the possible mechanisms of inactivation during the reaction (monomolecular inactivation of the free form of the enzyme, monomolecular inactivation of the enzyme-substrate complex, bimolecular inactivation of the enzyme by the substrate, bimolecular inactivation of the enzyme by the product):
(2.66)
Discrimination of inactivation mechanisms and determination of the kinetic characteristics of the reaction are usually carried out by several methods:
analyzing the dependence of product yield on enzyme concentration;
establishing a relationship between the degree of substrate conversion and the degree of enzyme inactivation;
carrying out the reaction at low degrees of substrate conversion and low enzyme concentrations;
carrying out the reaction at high concentrations of the enzyme;
preincubation of the enzyme with reaction components;
use of integral reaction equations.
The amount of enzyme present in tissues at any given time is determined by the relative rates of its synthesis and breakdown, as well as the concentrations of various types of inhibitors and activators. As a rule, the breakdown of enzymes and the decrease in their quantity in the medium occur slowly. Inhibition and activation of enzymes can be carried out quite quickly - within seconds.
There are many methods for determining and expressing the activity of individual enzymes. This is due to the diversity of enzymes, the presence and use of various substrates to determine their activity.
The International Biochemical Union has proposed the following definition of an enzyme unit: " A unit of any enzyme is taken to be the amount that catalyzes the conversion of one micromole of substrate per minute under given standard conditions ». The number of micromoles will be equal to the number of standard units . The International Commission suggested, if possible, that enzyme activity be determined at 30 °C and at pH values and substrate concentrations optimal for enzyme activity.
Are common properties of enzymes flow out from their protein nature . Enzymes thermolabile, their activity depends on pH and humidity , in which they operate, as well as from influence of activators and inhibitors .
When the temperature rises to certain limits, enzyme activity increases. When the temperature optimal for the enzyme is reached, its catalytic activity is at its highest. The optimal temperature for many enzymes is most often in within 40 to 50 °C (optimal for plant enzymes is 50 - 60 ° C, and for enzymes of animal origin - 40 - 50 ° C). However, the optimal temperature is not strictly constant and depends on many reasons, and in particular on the duration of heating. The longer the enzyme action, the lower the optimal temperature should be. .
In the temperature range from 0 to 50 °C, with an increase or decrease in temperature for every 10 °C, enzyme activity increases or decreases, respectively, by 1.4–2 times. With further heating, enzyme activity decreases, and at 80–100 °C enzymes usually completely lose their catalytic properties due to protein denaturation .
The temperature of inactivation (loss of activity) is different for different enzymes. Thus, inactivation of the enzyme amylase in solution occurs at 70 °C, sucrase - at 59, trypsin and pepsin - at 65 °C. In a dry state, enzymes can tolerate heating to higher temperatures. But at very high temperatures, enzyme inactivation occurs instantly. Pasteurization, sterilization, blanching and boiling destroy enzymes .
After thermal inactivation, some enzymes restore their catalytic activity. An example is peroxidase, which, even when heated for 60 s to 150 °C, does not completely lose its catalytic properties. Therefore, peroxidase is considered the most thermostable enzyme.
At temperatures below 0 °C, the catalytic activity of enzymes decreases sharply, but is still maintained even when food is frozen.
The reaction of the environment has a significant impact on the catalytic activity of enzymes. Enzymes change their solubility, osmotic pressure, viscosity and other properties under the influence of the pH of the environment. It is believed that changes in enzymatic activity depending on the pH of the environment are associated with changes ionization enzymes, substrate or enzyme-substrate complex .
Enzymes exhibit optimal activity only within certain pH limits inherent to them.. Thus, pepsin, which is released into the highly acidic environment of the stomach, has an optimum of activity at pH 1.5 and 2.5. At the same time, proteases, which are secreted by the pancreas into the duodenum, have optimal activity in the alkaline pH zone, and the optimum action of trypsin lies within the pH range of 8–9. At a pH value above or below the optimum, enzyme activity decreases .
Most enzymes are most active in neutral, slightly alkaline or slightly acidic environments. As the pH value shifts from optimal to acidic or alkaline, enzyme activity decreases.
Activators and inhibitors(paralyzers) of enzymes can accordingly strengthen or weaken and even stop their activity. Activators enzymes are metal ions: Na + , K + , Rb + , Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ and compounds containing sulfhydryl groups: SH, HCN, H 2 S . The presence of the specified metals or compounds in a solution in a certain concentration contributes to the manifestation of the full activity of some enzymes.
All enzymes are susceptible to inhibition due to denaturation or destruction of the enzyme protein.
The essence of the action of inhibitors in most cases is that they combine with active groups or active centers of the enzyme molecule. Distinguish general and specific inhibitors . TO general inhibitors which inhibit the action of all enzymes , include salts of heavy metals (lead, silver, mercury), trichloroacetic acid and tannin . Often the inhibition or cessation of the action of enzymes under the influence of heavy metals is reversible, and if substances that form compounds with these metals are added to the medium, the activity of the enzymes is restored.
Specific inhibitors act only on certain enzymes. Thus, hydrocyanic acid acts only on oxidative enzymes containing iron or copper in the active center. Hydrocyanic acid combines with metals, and the enzyme loses activity.
In a living cell, regulation of the action of enzymes is carried out not only with the help of specific activators and inhibitors, but also by binding enzymes to various colloidal structures of protoplasm. This binding of enzymes leads to their loss of activity. The release of the enzyme from the compound again restores its catalytic activity.
Enzymes inactivated at very high pressures . However, after the pressure is removed, the enzymes restore their catalytic activity.
The action of enzymes is greatly slowed down in dry foods, but does not stop completely. The results of enzyme activity can manifest themselves in changes in the quality of the product - its darkening, deterioration of aroma, taste, consistency, etc.
The rate of most enzymatic reactions is proportional to the concentration of the enzyme, at least in the earliest stages. Beyond the initial stages, the rate of enzymatic reactions decreases.
The enzyme forms a complex with the substrate, which dissociates into the free enzyme and the final reaction product:
where E is the enzyme; S – substrate; ES – enzyme-substrate complex; P – final product.
The amount of substrate is very large compared to the amount of enzyme, and therefore the concentration of the substrate greatly influences the rate of enzymatic reactions. If the substrate is contained in significant excess, then the amount of product formed is proportional to time. As the substrate concentration decreases, the amount of the final product (P) formed per unit time decreases.
The presence of an enzyme in a solution is judged by its action. Thus, the presence of amylase in saliva can be judged by the ability of saliva to saccharify starch, the presence of gastric pepsin - by its ability to dissolve egg white or fibrin with sufficient speed.
By regulating the activity of enzymes by creating an appropriate reaction environment, you can control the speed of the reactions they catalyze, as well as the activity of enzymes contained in food products, which allows you to carry out measures for the storage of grain, potatoes, fruits and vegetables, the production of a number of products (wine, tea, etc. .).
Nomenclature and classification of enzymes
In the initial period of development of the study of enzymes, they were given names without a specific system, based on random characteristics, the name of the substrate or the type of reaction catalyzed. So, the enzyme pepsin got its name from the Greek word “pepsis” - I digest, papain - from the juice of the papaya plant, rich in the enzyme. It happened that individual authors gave different names to the same enzyme.
In connection with the rapid development of the science of enzymes - fermentology, in 1961, the standing committee on enzymes at the International Biochemical Union developed a modern nomenclature and classification of enzymes. In accordance with this classification, the name of the enzyme was composed of the chemical name of the substrate and the name of the reaction that was carried out by the enzyme. To the Latin name of the root of the substrate on which the enzyme acts (sucrose - sucrase), or to the name of the process catalyzed by this enzyme (hydrolysis - hydrolases), ending added"aza". Along with new names for many enzymes, old ones that have become firmly established in the scientific literature (pepsin, trypsin, papain, etc.) have been preserved.
According to modern classification, all enzymes are divided into six classes: oxidoreductases; transferases; hydrolases; lyases; isomerases; ligases (synthetases) . The classification of enzymes is based on the nature of their action.
Each class is divided into subclasses, and each subclass is divided into groups.
Oxidoreductases
These are enzymes that catalyze redox reactions that occur in living organisms. Oxidation reactions of substances in organisms are always accompanied by reduction reactions. Oxidoreductases are divided into 14 subclasses (the most extensive class of enzymes).
Oxidation occurs as the process of removing hydrogen (electrons) from the substrate, and reduction occurs as the addition of hydrogen atoms (electrons) to the acceptor. This reaction can be schematically represented as follows:
AN 2 + B = A + VN 2,
where AN 2 is a substance that donates its hydrogen and is called a donor; B is a substance that takes away hydrogen and is called an acceptor.
A variety of substances can undergo oxidation - carbohydrates, fats, proteins, amino acids, vitamins, etc.
The role of oxidoreductases in living tissues is performed by extensive groups dehydrogenases And oxidases , which are named depending on the substrate they oxidize. Thus, the enzyme that dehydrates malic acid is called malate dehydrogenase, the enzyme that dehydrogenates ethyl alcohol is called alcohol dehydrogenase, etc.
In the class of oxidoreductases, the main ones are dehydrogenases, which carry out the dehydrogenation reaction. All dehydrogenases are divided into two groups : anaerobic and aerobic, which are called oxidases .
Anaerobic dehydrogenases are specific enzymes that catalyze hydrogen abstraction from certain chemicals and transmitting it to other enzymes - hydrogen carriers. These dehydrogenases are two-component enzymes in which the coenzyme is easily separated from the protein part. As a coenzyme Anaerobic dehydrogenases may contain two substances - nicotine amide adenine nucleotide ( ABOVE ) or nicotine amide adeline nucleotide phosphate ( NADP ). Both of these substances have exceptionally high reactive redox properties.
There are many known anaerobic dehydrogenases that catalyze the oxidation of various organic compounds. Thus, lactate dehydrogenase catalyzes the oxidation of lactic acid to pyruvic acid, isocitrate dehydrogenase - the oxidation of isocitric acid to oxalic-succinic acid.
To the group aerobic dehydrogenases (oxidases) include enzymes that contain as a coenzyme included vitamin B 2 , (riboflavin ), therefore such enzymes are called flavin . Flavin enzymes are capable of removing hydrogen from the substance being oxidized and transferring it to other compounds or air oxygen:
2H 2 O 2 → 2H 2 O + O 2.
Taking hydrogen from the substance being oxidized and transferring it to air oxygen, the oxidase can form water or hydrogen peroxide (H 2 O or H 2 O 2). This group of enzymes includes polyphenol oxidase, ascorbate oxidase, and glucose oxidase.
Polyphenol oxidase is an aerobic dehydrogenase for which The hydrogen acceptor is oxygen gas .
It acts on o-diphenols, polyphenols, tannins and tyrosine. Polyphenol oxidase is widely distributed in fungi and higher plants, especially in green tea leaves. The action of polyphenol oxidase explains the darkening of the cut flesh of fruits and vegetables, potatoes, as well as the darkening of fresh tea leaves when rolled. Polyphenol oxidase plays an important role as an intermediate in plant respiration.
Enzyme peroxidase along with polyphenol oxidase and cytochrome oxidase, it actively participates in plant respiration processes and plant defense reactions against plant pathogenic microorganisms.
The active group of peroxidase contains iron . Using the enzyme peroxidase due to hydrogen peroxide and some other organic peroxides, oxidation of organic compounds occurs. Peroxidase forms a complex organic compound, as a result of which the peroxide is activated and acquires the ability to act as a hydrogen acceptor:
Many organic compounds react with atmospheric oxygen and form peroxides. Peroxides are especially easily formed when compounds with unsaturated bonds are oxidized by atmospheric oxygen: carotenoids, unsaturated fatty acids, and some hydrocarbons.
Enzyme catalase catalyzes the process of splitting hydrogen peroxide into water and oxygen:
The catalase molecule, like peroxidase, contains iron . The main purpose of catalase in the body is that it destroys hydrogen peroxide, which is harmful to cells, formed during respiration.
Enzyme lipoxygenase catalyzes the formation of peroxides and hydroperoxides during oxidative spoilage of fats.
The rate of an enzymatic reaction, in other words, the activity of the enzyme is also determined by the presence of activators and inhibitors in the medium: the former increase the reaction rate and sometimes modify it, the latter inhibit the reaction. Among the chemical compounds that affect the activity of enzymes, there are various substances. Thus, HC1 activates the action of pepsin, bile acids - pancreatic lipase; some tissue enzymes (oxidoreductases, cathepsins, arginase), plant proteinase papain, etc. are significantly activated by compounds containing free SH groups (glutathione, cysteine), and some also by vitamin C. Ions especially often serve as activators divalent and sometimes monovalent metals. Many enzymes are not active at all in the absence of metals. Thus, when removing zinc, carbonic anhydrase is practically devoid of enzymatic activity; Moreover, during the action of this enzyme, zinc cannot be replaced by any other metal. Enzymes are known whose action is activated by a number of metals, in particular, enolase (see Carbohydrate metabolism) is activated by Mg 2+, Mn 2+, K +. In table 18 shows examples of the participation of metals in the action of some enzymes.
| Table 18. Metals in the activation of certain enzymes 1 (1 It is usually difficult to draw the line between metalloenzymes (the metal is complexly bound and irreplaceable) and enzymes activated by metals (the latter only speed up the reaction and easily dissociate).) | |||
| Enzyme | Metal | Enzyme | Metal |
| Cytochromes | Fe | Amylase | Sa |
| Catalase | Fe | Lipase | Sa |
| Peroxidase | Fe | Carbonic anhydrase | Zn |
| Tryptophan oxidase | Fe | Lactate dehydrogenase | Zn |
| Homogentisicase | Fe | Uricaza | Zn |
| Ascorbate oxidase | Si | Carboxypeptidase | Zn |
| Tyrosinase | Si | Peptidases | Mg |
| Phenoloxidase | Si | Phosphatases | Mg |
| Xanthine oxidase | Mo | Phosphoglucokinase | Mg |
| Nitrate reductase | Mo | Arginase | Mn |
| Aldehyde oxidase | Mo | Phosphoglucomutase | Mn |
| Peptidases | Co | Cholinesterase | Mn |
Regarding the role of metals in the activating action of enzymes, available data indicate that in some cases metal ions (Co 2+, Mg 2+, Zn 2+, Fe 2+) perform the functions of prosthetic groups of enzymes. In other cases, they contribute to the attachment of the substrate to the active site and the formation of an enzyme-substrate complex. For example, Mg 2+ ions, through a negatively charged phosphate group, ensure the addition of monophosphoric esters of organic substances to the active center of phosphatases that catalyze the hydrolysis of these compounds. In some cases, the metal combines with the substrate, forming the true substrate on which the enzyme acts. In particular, Mg 2+ ions activate creatine phosphokinase due to the formation of the true substrate and the magnesium salt of ATP. Finally, there is experimental evidence of the direct participation of metals (for example, Ca 2+ ions in the salivary amylase molecule) in the formation and stabilization of the active center and the entire tertiary structure of the enzyme molecule. It should also be noted that metals often play a role as allosteric modulators (see Fig. 59). By interacting with the allosteric center, such a metal (modulator) promotes the formation of the most favorable spatial configuration of the enzyme and the active enzyme-substrate complex.
Anions at physiological concentrations are usually ineffective or have little activating effect on enzymes. The exceptions are pepsin, some oxidoreductases activated by anions, as well as salivary amylase, which catalyzes the hydrolysis of starch, the activity of which is increased by chlorine ions, and adenylate cyclase, which is activated by halogen anions.
Inhibitors It is customary to call substances that cause partial or complete inhibition of reactions catalyzed by enzymes. Since enzymes are proteins, any agents that cause protein denaturation (heat, acids, alkalis, heavy metal salts) lead to inactivation of the enzyme. However, such inactivation is relatively nonspecific. It is not related to the mechanism of action of enzymes. A much larger group consists of so-called specific inhibitors, which act on one enzyme or a group of related enzymes. Research into these inhibitors is important for a number of reasons.
First, inhibitors can provide valuable information about the nature of the active site of the enzyme, as well as its functional groups and chemical bonds that ensure the formation of the enzyme-substrate complex. Substances are known that specifically bind one or another group in an enzyme molecule, excluding it from the sphere of a chemical reaction. In particular, iodoacetate ICH 2 -COOH, its amide and ethyl ester, parachloromercuribenzoate ClHg - C 6 H 4 -COOH and other reagents relatively easily enter into chemical bonds with some SH groups of enzymes. If such groups are essential for the act of catalysis, then the addition of such inhibitors leads to a complete loss of enzyme activity:
R-SH + ICH 2 -COOH --> HI + R-S-CH 2 -COOH
A number of other enzymes (cholinesterase, trypsin and chymotrypsin) are strongly inhibited by certain organophosphorus compounds, in particular diisopropyl fluorophosphate (DFP), due to blocking of the key hydroxyl group of serine in the active site (see above).
Secondly, inhibitors have found wide use in enzymology in studying the nature of multiple forms of enzymes and isoenzymes that differ not so much in electrophoretic mobility as in the difference in reactions to the same inhibitor.
With the help of inhibitors that selectively turn off individual stages of a multi-stage metabolic process, the sequence of chemical reactions and the nature of the enzymes involved can be accurately determined. In particular, in this way, with the use of iodoacetate, fluoride and other inhibitors, the glycolytic pathway of redox transformations of glucose to lactic acid in muscle tissue was deciphered (see Carbohydrate metabolism), which has 11 stages involving 11 enzymes and 10 intermediate metabolites.
The mechanism of action of many toxins and poisons on the body is based on enzyme inhibition. Thus, it is known that in cases of hydrocyanic acid poisoning, death occurs due to complete inhibition of respiratory enzymes (cytochrome oxidase), especially brain cells. The toxic effect of some insecticides on the human and animal body is due to inhibition of the activity of cholinesterase, an enzyme that plays a primary role in the activity of the nervous system.
Rational chemotherapy - the conscious use of drugs in medicine, should be based on an accurate knowledge of the mechanism of their action, the biosynthesis of enzymes or their work in the body. Sometimes the treatment of human diseases involves the use of selective inhibitors. Thus, the inhibitor of trypsin, chymotrypsin and kallikrein, trasylol, is widely used in the treatment of acute pancreatitis. The selective inhibitory effect on enzymes of certain natural and synthetic compounds (so-called antimetabolites) currently serves as the basis for the development of effective methods for the synthesis of chemotherapeutic drugs. This path opens up wide possibilities for regulating both the synthesis of enzymes and the intensity of metabolism.
Types of inhibition. Although the mechanism of action of most inhibitors is unclear, a distinction is usually made between reversible and irreversible inhibition. If an inhibitor molecule causes permanent changes or modification of the functional groups of the enzyme, then this type of inhibition is called irreversible. More often, however, reversible inhibition occurs, which can be quantitatively studied based on the Michaelis-Menten equation. Reversible inhibition, in turn, is divided into competitive and non-competitive, depending on whether it is possible or not to overcome the inhibition of the enzymatic reaction by increasing the concentration of the substrate. In the second case, increasing the substrate concentration does not change the degree of enzyme inhibition.
Competitive inhibition can be caused by substances that have a structure similar to the substrate, but slightly different from the structure of the true substrate. A classic example of this type of inhibition is the inhibition of succinate dehydrogenase activity by malonic acid. This enzyme catalyzes oxidation by dehydrogenating succinic acid to fumaric acid according to the scheme:
If malonic acid (inhibitor) is added to the medium, then due to its structural similarity with the true substrate succinic acid (the presence of two of the same ionized carboxyl groups), it will react with the active center to form an enzyme-inhibitor complex (see diagram), however, In this case, hydrogen transfer from malonate does not occur. Since the structures of the substrate - succinic acid and the inhibitor - malonate are still somewhat different, they compete for binding to the active site, and the degree of inhibition will be determined by the ratio of the concentrations of malonate and succinate, and not by the absolute concentration of the inhibitor. This type of inhibition is sometimes called metabolic antagonism inhibition (Fig. 56).
In general form, the reaction between an inhibitor and an enzyme can be represented by the following equation:
The resulting complex, called the enzyme-inhibitor complex (EI), unlike ES, does not decompose to form reaction products. The dissociation constant of the EI complex or inhibitory constant (K 1) can, following the Michaelis-Menten theory, be determined as the ratio of the constants of the reverse and forward reactions:
i.e., the inhibitory constant is directly proportional to the product of the concentration of the enzyme and inhibitor and inversely proportional to the concentration of the EI complex.
The method of competitive inhibition has found wide application in medical practice. It is known, for example, that sulfonamide drugs are used to treat certain infectious diseases caused by bacteria. It turned out that these drugs are structurally similar to para-aminobenzoic acid, which the bacterial cell uses to synthesize folic acid, which is an integral part of bacterial enzymes. Due to this structural similarity, sulfonamide, for example, blocks the action of the enzyme by displacing para-aminobenzoic acid from the complex with the enzyme that synthesizes folic acid, which leads to inhibition of bacterial growth.
Some analogues of vitamin B6 and folic acid, in particular deoxypyridoxine and aminopterin (see Vitamins), act as competitive, so-called coenzyme inhibitors (or antivitamins), inhibiting many biochemical processes in the body.
Non-competitive inhibition is caused by substances that have no structural similarity to substrates and often bind not to the active center, but elsewhere in the enzyme molecule. The degree of inhibition in many cases is determined by the duration of action of the inhibitor on the enzyme. With this type of inhibition, due to the formation of a stable covalent bond, the enzyme often undergoes complete inactivation, and then the inhibition becomes irreversible. Examples of non-competitive inhibition (inactivation) are the action of iodoacetate, diisopropyl fluorophosphate, as well as diethyl-n-nitrophenyl phosphate and hydrocyanic acid, which consists in binding and switching off functional groups or metal ions in the enzyme molecule.
To clarify the question of the type of inhibition, use the Michaelis-Menten equation, the Lineweaver-Burk graph and other more advanced equations, for example the Edie-Hofstee equation:
v = - K m (0/[S]) + V max
and corresponding graphs in rectilinear coordinates. In the graphs below, plotted in coordinates v and [S], as well as in coordinates 1/v and 1/[S], V is the maximum reaction rate, V 1 is the maximum rate in the presence of an inhibitor, K 1 is the inhibitory constant; all other symbols have been presented above.
It can be seen that with a competitive type of inhibition (Fig. 57), the inhibitor increases the value of K m (by an amount equal to the difference in the length of the segments cut off from the x-axis), without affecting the maximum speed. This means that at a sufficiently high substrate concentration [S], the inhibitor is displaced by substrate molecules from the EI complex. With non-competitive inhibition (Fig. 58), the inhibitor reduces the maximum speed. If the value of K m does not decrease, then we speak of completely non-competitive inhibition. A similar type of inhibition occurs during the formation of inactive, difficult to dissociate EI and (or) EIS complexes. Often, however, there is a mixed type of inhibition (sometimes called the partially noncompetitive type), in which a decrease in Vmax is combined with an increase in Km. This means that the EI complex retains partial activity, i.e., the ability to form an intermediate ternary complex EIS, in which the substrate undergoes a delayed catalytic transformation. In rare cases, the degree of inhibition of enzyme activity may increase with increasing substrate concentration; For this type of inhibition, the rather inaccurate term non-competitive (from the English uncompetitive) was proposed. One of the mechanisms of such inhibition is due to the possibility of combining the inhibitor with the ES complex to form an inactive or slowly reacting ternary ESI complex.
Thus, by graphically analyzing enzymatic reaction rates as a function of substrate concentrations, valuable information on the kinetics of enzymatic reactions can be obtained, illuminating the possible mechanism of enzymatic catalysis.
Regulation of enzyme activity
It was stated above that one of the unique properties of living organisms is the amazing ability to balance catabolic (biodegradative) and anabolic (biosynthetic) processes. Although the processes of synthesis, decomposition and interconversion of hundreds and thousands of various substances simultaneously take place in cells, there are many regulatory mechanisms that ensure the constancy of the internal environment of the body. Some of these regulatory mechanisms, among which an important role is played by mechanisms regulating enzyme activity, will be discussed below.
Influence of the law of mass action. In a reversible chemical reaction catalyzed by an enzyme, for example A + B C + D, the concentration of the reaction components and, accordingly, the direction of the reaction will be regulated by the influence of the law of mass action. In particular, it can be shown in the reversible transamination reaction catalyzed by alanine aminotransferase:
Alanine + α-Ketoglutarate Pyruvate + Glutamate.
This type of regulation obviously plays only a limited role, since in real conditions the reaction usually proceeds in one direction, since the resulting products may turn out to be substrates for the action of other enzymes and are removed from the reaction; in these cases, a stable (stationary) state is established rather than a true equilibrium.
Change in the amount of enzyme. In bacteria, the phenomenon of induced synthesis of enzymes has been well studied when they are grown in a medium where the only source of carbon and energy is one or another carbohydrate, for example, glucose. Replacing glucose in the medium with lactose leads to the induced or adapted (after a short period of lag phase) synthesis of the enzyme galactosidase (programmed by the lactose gene, see Protein Synthesis), which breaks down lactose into glucose and galactose. In animal tissues, such rapid synthesis of enzymes is observed relatively less frequently, and the mechanism inducing synthesis has been studied only for a small number of enzymes (tyrosine transaminases, serine and threonine dehydratases, tryptophan pyrrolase, etc.). However, when certain poisons, carcinogenic substances, alkaloids, insecticides, etc. enter the body, a sharp increase in the activity (respectively, the quantity) of enzymes is observed after a few days - hydroxylases of the smooth endoplasmic reticulum of liver cells; oxidizing foreign substances into products that are non-toxic to the body. On the other hand, cases have been described when, under the action of such hydroxylases, foreign substances are converted in the body into more toxic compounds. This phenomenon, the opposite of detoxification, is called lethal synthesis.
Proenzymes. Proteolytic enzymes of the gastrointestinal tract and pancreas are synthesized in an inactive form, in the form of proenzymes (zymogens). Regulation in these cases comes down to the conversion of proenzymes into active enzymes under the influence of specific agents. Thus, trypsin is synthesized in the pancreas in the form of trypsinogen. The latter is converted into active trypsin in the intestine under the action of another protein enzyme - enterokinase, first discovered in the laboratory of I. P. Pavlov. It has been established that the activating effect of enterokinase is reduced to the cleavage of a hexapeptide from trypsinogen, leading to the formation of the native tertiary structure of trypsin and its active center (see above); autocatalysis is also observed. The conversion of inactive pepsinogen into active pepsin occurs autocatalytically as a result of limited proteolysis in the presence of HC1 and is also associated with cleavage from the first specific inhibitor of a polypeptide nature (see Metabolism of simple proteins). The synthesis of proteinases in an inactive form and a number of other inactive precursor proteins obviously has a certain biological meaning, preventing the destruction of organ cells in which proenzymes are formed.
Chemical modification of the enzyme. It was indicated above (see Protein Chemistry) that a number of proteins undergo postsynthetic modification during the formation of their tertiary structure. It turned out that the key enzymes of energy metabolism - phosphorylase, glycogen synthetase, etc. - are also controlled by phosphorylation and dephosphorylation carried out by specific enzymes - protein kinase and protein phosphatase, the level of activity of which is in turn regulated by hormones (see Carbohydrate metabolism). The level of activity of key enzymes and, accordingly, the intensity of metabolic processes will be determined by the ratio of phosphorylated and dephosphorylated forms of these enzymes.
Regulation of enzyme activity according to the feedback principle. In many strictly biosynthetic reactions, the main type of rate regulation of a multistep enzymatic process is feedback inhibition, when the final product of the biosynthetic chain suppresses the activity of the enzyme catalyzing the first step.
Let us assume that a multi-stage biosynthetic process takes place in cells, each stage of which is catalyzed by its own enzyme:
The rate of such a total sequence of reactions is largely determined by the concentration of the final product (P), the accumulation of which above the permissible level has a powerful inhibitory effect on the first stage of the process, respectively, on the enzyme E 1.
The existence of such a mechanism for controlling enzyme activity by metabolites was first demonstrated in E. coli when studying the synthesis of isoleucine and cytidine triphosphate (CTP). It turned out that isoleucine, which is the final product, selectively suppresses the activity of threonine dehydratase, which catalyzes the first step in the process of converting threonine into isoleucine, which includes five enzymatic reactions. Similarly, CTP, as the end product of the biosynthetic pathway, has an inhibitory effect on the first enzyme (aspartate transcarbamoylase), thereby regulating its own synthesis. This type of inhibition is called feedback inhibition or retroinhibition. Its existence has been proven in all living organisms, and currently it is considered as one of the leading types of regulation of enzyme activity and cellular metabolism in general 1. (1 It should be pointed out that the reaction rate (as well as enzyme activity) in purely biodegradative (catabolic) processes is regulated by intermediate products that are indicators of the energy state of the cell (purine nucleotides, pyrophosphate, inorganic phosphate, etc.).)
On the other hand, in amphibolic processes (see Introduction to metabolism and energy), which simultaneously perform biosynthetic and biodegradative functions 2, the existence of regulation has been proven both by the type of retroinhibition and by macroergs - indicators of the energy state of the cell. (2 Amphibolic processes include pathways such as glycolysis, glycogenolysis, tricarboxylic acid cycle, hexose monophosphate pathway, transamination of amino acids (see Metabolism)). For amphibolic processes, a unique type of regulation, peculiar only to them, is, in addition, activation by a precursor, when the first metabolite in a multistep pathway activates the enzyme that catalyzes the last stage. Thus, the activating effect of glucose-6-phosphate, which is a precursor of glycogen, on the enzyme glycogen synthetase has been proven.
Similar types of inhibition by the final product and activation by the first product are characteristic of allosteric (regulatory) enzymes (see above), when the effector, structurally different from the substrate, binds to a special (allosteric) center of the enzyme molecule, spatially distant from the active center. Therefore, it is common to distinguish between the allosteric type of regulation, which includes both allosteric inhibition and allosteric activation. Interconversions of active and inactive allosteric enzymes in a simplified form, as well as conformational changes observed upon attachment of the substrate and effectors, are presented in Fig. 59.
It can be seen that the attachment of a negative effector to the allosteric center causes significant changes in the configuration of the active center of the enzyme molecule and, as a result, loss of the enzyme’s affinity for its substrate (formation of an inactive complex).
Allosteric interactions are manifested in the nature of the curves of the dependence of the initial reaction rate on the concentration of the substrate or effector, in particular in the S-shape of the curves (deviation from the hyperbolic Michaelis-Menten curve). This means that the binding of one substrate molecule facilitates the binding of a second molecule at the allosteric center, thereby increasing the rate of the reaction. In addition, regulatory (allosteric) enzymes are characterized by a nonlinear dependence of the reaction rate on the enzyme concentration.
Other types of regulation of enzyme activity. There are a number of other mechanisms that control the rate of metabolic processes and the activity of intracellular enzymes. Such mechanisms may include competition between enzymes for a common substrate, shutdown of the activity of one of the enzymes (in multiple forms of enzymes), the influence of cofactor concentrations and their form (especially metal ions), and the phenomenon of compartmentalization. The compartmentalization mechanism apparently plays an important biological role, spatially separating enzymes from their substrates through biomembranes (for example, lysosomal enzymes: proteinases, phosphatases, ribonucleases and other hydrolytic enzymes, from substances on which they act in the cytoplasm) or mutually incompatible in metabolic processes at the same time. An example of the latter may be the pathways for the synthesis of fatty acids, which occur mainly in the soluble fraction of the cytoplasm, and the pathways for the breakdown of fatty acids, concentrated in the mitochondria.
Determination of enzyme activity
Determining the quantitative content of enzymes in biological objects presents certain difficulties, since, with rare exceptions, enzymes in tissues are present in negligibly small concentrations. Therefore, the amount of enzymes is judged by the rate of the catalyzed reaction, under certain agreed measurement conditions. Under optimal conditions of temperature, pH of the environment and complete saturation of the enzyme with the substrate, the rate is proportional to the concentration of the enzyme. The rate of an enzymatic reaction is judged either by the rate of loss of the substrate or by the rate of formation of the reaction product.
To express the concentration of an enzyme, the Commission on Enzymes of the International Biochemical Union recommends a standard unit (E). A unit of any enzyme is taken to be the amount that, under optimal conditions, catalyzes the conversion of 1 µmol of substrate per minute (µmol/min). A new definition of the international unit of enzyme catal (kat) has been proposed, corresponding to the amount of enzyme capable of causing the conversion of 1 mole of substrate into a product in 1 s (1 mol/s). The relationship of the international unit (E) to the katal can be expressed as follows:
or 1 E=1 µmol · min -1 = (1/60) µmol · s -1 = (1/60) µkat = 16.67 nkat. Thus, 1E of the enzyme corresponds to 16.67 ncat.
It is also recommended to measure enzyme units at 25°C, the optimum pH and substrate concentration above the saturation concentration. In these cases, the rate corresponds to a zero-order reaction with respect to the substrate and will depend only on the concentration of the enzyme.
To express the activity of an enzyme, the definition of specific and molecular activity is used. The specific activity of an enzyme is usually expressed as the number of units of enzymatic activity per 1 mg of protein (or the number of catals per 1 kg of active protein). The number of substrate molecules that are converted by one enzyme molecule per minute is usually called the number of revolutions, or molecular activity. Thus, one molecule of erythrocyte catalase is capable of breaking down 5 · 10 6 molecules of hydrogen peroxide 1 in 1 minute. (1 For 1 atom of inorganic iron, which also catalyzes the decomposition of H 2 O 2 , to break down the number of H 2 O 2 molecules that catalase breaks down in 1 s, it would take more than 300 years. This example is clear evidence of one of the main properties of enzymes - their high catalytic activity.)
Teacher:Ph.D.
Kuznetsova Ekaterina Igorevna Mechanisms of enzyme inactivation
1. Change of primary structure:
1.1. Polypeptide chain rupture:
Severe conditions (prolonged boiling in HCl) –
hydrolysis to individual amino acids.
When heated to 100 °C (pH 7-8), hydrolysis
peptide bonds are insignificant.
Most sensitive to
high temperature hydrolysis are
peptide bonds formed by residues
aspartic acid.
Proteases (bacterial contamination, autolysis). Solution:
inactivated enzymes. 1.2.Oxidation of enzyme functional groups
SH groups of cysteine and indole fragments
tryptophan, at elevated temperatures, can
oxidize (sulfoxy-cysteine compounds
(SOH, SO2H) and products are formed
opening of the indole ring of tryptophan. Solution:
Reactivate with restoratives
agents, in particular low molecular weight thiols
(for example, cysteine or dithiothreitol).
Caused by: Thiols and other reduced
sulfur compounds, for example Na2SO3, Na2S2O3.
Disulfide bond reduction product
(S-S) is:
1) thiol form (protein–SH)
2) mixed disulfide of the thiol form of the protein with
a reducing reagent, for example
protein–S–SO3). 1.3. Cleavage of disulfide bonds
Alkaline hydrolysis of cysteine →dehydroalanine→
Due to its nucleophilic properties
interacts with NH2 groups of lysine and SHcysteine →lysinoalanine and lanthionine.
For complete destruction of all S–S bonds, fairly stringent conditions are required (0.1–1 M alkali,
100 °C).
However, the destruction of the most reactive
S–S bonds can occur in fairly soft
conditions - for example, at temperatures of 60–80 ° C and
slightly alkaline pH values.
Should be taken into account when using enzymes in
as additives to detergents. Solution:
Addition of thiols to the medium will lead to
cleavage of mixed disulfide and
subsequent formation of the correct S–S bond 1.4. Chemical modification of catalytic SH groups.
Heavy metal cations (Hg, Pb and Cu)
bind to the SH groups of the active site
enzyme
↓
Formation of the corresponding mercaptides
↓
The enzyme is inactivated
10.
1.5. Phosphorylation of proteins in vivo.Under the influence of phosphorylase and phosphatase,
contained in semi-purified enzymatic
drugs in the form of impurities
↓
Phosphoric acid binds to OH groups
serine and threonine.
↓
Conformational changes in protein
molecule
↓
enzyme inactivation.
11.
Solution:There are practically no examples in the literature
good luck reactivating this way
inactivated enzymes.
12.
1.6. Deamination of asparagine residues.At temperatures (about 100 °C) and pH (about
4.0–5.0) deamination of residues occurs
asparagine.
↓
enzyme inactivation.
13.
Solution:There are practically no examples in the literature
good luck reactivating this way
inactivated enzymes.
14.
1.7. Radiation inactivation of enzymesγ-irradiation and UV light
Affect functional groups
enzymes, peptide bonds and SH groups
cysteine residues.
15.
2. AggregationObserved at elevated temperatures, at
extreme pH values, in the presence
some chemical compounds.
The higher the concentration, the faster it goes
aggregation.
Hydrophobic interactions and hydrogen
bonds, the formation of disulfide bonds is possible
bridges between individual proteins
molecules
16.
Solution:It is necessary to destroy intermolecular
covalent and non-covalent contacts c
using concentrated solutions
urea and guanidine chloride, extreme
pH values.
If the aggregation of enzymes results in the formation of
intermolecular S-S bridges are added to the medium
relatively low concentrations
(μmol/L) thiol-containing reagents (e.g.
cysteine or dithiothreitol).
At such concentrations, intramolecular
S–S bonds in the protein are usually not affected.
17.
3. Inactivation of enzymes by surfacetension
Surface tension at the interface
between air and clean water is 80
din/cm.
Foaming causes denaturation
enzymes adsorbed at the interface
phases
18.
Solution:Adding a surfactant reduces the surface
tension up to 1 dyne/cm.
19.
4. Sorption of protein on the walls of the reactionvessel
Sorption due to non-covalent interactions
leads to a decrease in enzyme concentration in
solution.
Must be taken into account when working with
diluted protein solutions
(concentration 10-8–10-10 mol/l).
Under the influence of denaturing factors
ability of proteins to adsorb on walls
reaction vessel may increase.
20.
Solution:Desorption of enzyme from the walls of the reaction
the vessel is achieved through destruction
nonspecific interactions between
protein and sorption centers on the surface
vessel.
Extreme pH values can be used
concentrated solutions of urea or
guanidine chloride.
21.
5. Dissociation of oligomeric proteins intosubunits
Caused by: Urea, detergents, acids or
heating.
Lead to:
conformational changes of individual
subunits;
subunit aggregation;
dissociation of cofactors from active centers;
modifications of functional groups that
oligomeric protein were shielded from
contact with solvent.
22.
6. Desorption of cofactor from the active siteenzyme
Causes: heating, chelating effects, dialysis
If cofactor dissociation is accompanied
significant conformational shifts or
chemical modification of important
functional groups → enzyme
is irreversibly inactivated.
If no significant
changes in protein conformation, then adding
in an environment of excess cofactor leads to
enzyme reactivation.
23.
Cofactor regenerationRegeneration methods:
Enzymatic (methods using conjugated substrates or enzymes)
Non-enzymatic (chemical and
electrochemical approaches)
24.
Enzymatic methodExcessive amounts are introduced into the system.
conjugate substrate of the same enzyme:
Example: when alcohol dehydrogenase works
NADH is consumed.
25.
Enzymatic method1. Use of conjugated substrates.
Disadvantage:
high concentrations are used
conjugated substrate, since the equilibrium
reactions are greatly shifted to the side
alcohol formation;
complicates the procedure for identifying the main
product from the reaction mixture.
26.
Enzymatic method2. Use of paired
enzymatic reactions
Enzyme 2 is additionally introduced into the system,
whose functioning is ensured
coenzyme regeneration.
The enzymes used in the system must have
different substrate specificity
27.
Non-enzymatic methods1. Chemical methods.
Sodium dithionite and some
pyridinium salts:
+ Low cost.
- can inhibit certain enzymes.
Flavin coenzymes
28.
Non-enzymatic methods2. Electrochemical methods.
Direct electrochemical reduction or
oxidation.
“-” appearance during the regeneration process
enzymatically inactive forms of coenzyme,
for example, as a result of its dimerization.
29. STABILIZATION OF ENZYMES IN BIOTECHNOLOGICAL SYSTEMS
30.
Problems encountered during useenzymes in biotechnological processes:
1. Elevated temperatures
2. Extreme pH values
3. High concentrations of organic
solvents or surfactants.
4. Inability to reuse
enzyme.
5. Difficulty in separating the enzyme from
product.
31.
Basic approaches for stabilizationenzymes:
1. Adding stabilizing substances to the medium,
in which the enzyme is stored or carried out
enzymatic reaction.
2. Chemical modification of the enzyme.
3. Immobilization of the enzyme.
32.
1. Substrates or their analogues:
The enzyme-substrate complex is often more
more stable than the free enzyme.
Example: Lactate dehydrogenase in the presence
lactate is more heat stable.
33.
Enzyme stabilization using:2. Organic solvents:
Polyhydric alcohols stabilize some
enzymes by increasing stability
intramolecular protein hydrogen bonds.
Example: Chymotrypsin in the presence of 50–90%
glycerol is more resistant to proteolysis
34.
Enzyme stabilization using:3. Soleil:
At low salt concentrations (<0,1M) катионы
Ca2+, Zn2+, Mn2+, Fe2+, etc. can specifically
interact with metalloproteins.
Some of them are cofactors.
Ca2+ is capable of stabilizing tertiary
the structure of a number of proteins due to the formation
ionic bonds with two different
amino acid residues.
Example: α-Amylase (from bacillus caldolyticus)
Ca2+ significantly increases thermal
sustainability.
35.
36.
Chemical modification of the enzyme1. The enzyme takes on a more stable
conformation.
2. Introduction of new functional groups into the protein
leads to the formation of additional
stabilizing hydrogen bonds or salt
bridges.
3. When using non-polar compounds
hydrophobic interactions are enhanced.
4. Modification of hydrophobic surface areas
protein by hydrophilic compounds reduces
area of unfavorable contact of external
non-polar residues with water.
Example: glutaraldehyde
37.
Enzyme immobilization allows:Increase enzyme stability (heat,
autolysis, exposure to aggressive environments, etc.)
1. Reuse the enzyme
2. Separate enzyme from reagents and products
reactions.
3. Interrupt the reaction at the right moment.
38.
Immobilized enzymes are drugsenzymes whose molecules are associated with
carrier, while retaining completely or
partly its catalytic properties.
Immobilization methods:
1. Chemical
2. Physical
39.
Immobilization methods:The following media can be used:
1)Organic materials:
1.1) natural (polysaccharides, proteins, lipids)
1.2) synthetic polymer carriers
2) Inorganic materials (matrices on
based on silica gel, clay, ceramics, natural
minerals, etc.)
40.
1) adsorption of the enzyme on an insoluble carrier
as a result of electrostatic, hydrophobic,
van der Waals and other interactions;
;
structures;
4) Connection to a two-phase system.
41.
Methods of physical immobilization:Achieved by contact of an aqueous solution
enzyme with a carrier.
42.
Methods of physical immobilization:1) adsorption of the enzyme on an insoluble carrier
Factors influencing adsorption:
1. Specific surface area and porosity of the carrier
2. pH value (on non-ion exchangers max adsorption
at the isoelectric point of the protein)
3. Ionic strength of the solution (increasing ionic strength –
enzyme desorption, but sometimes the opposite situation
“salting out”)
4. Enzyme concentration.
5. Temperature (on the one hand denaturation, on the other
another accelerated diffusion)
43.
Methods of physical immobilization:1) adsorption of the enzyme on an insoluble carrier
Advantages:
2) Media availability
Flaws:
1) Insufficient bonding strength
2) Many media are biodegradable
44.
Methods of physical immobilization:2) inclusion of the enzyme in the semipermeable
capsule, in a semi-permeable membrane
45.
Methods of physical immobilization:2) inclusion of the enzyme in the semipermeable
capsule, in a semi-permeable membrane
Advantages:
1) Relative simplicity of the technique
2) Protection against microorganisms
3) There are no diffusion restrictions (since
The surface to area ratio is high and
membrane thickness is small)
Flaws:
1) Biodegradable
2) Not applicable for high molecular weight
46.
Methods of physical immobilization:3) mechanical inclusion of the enzyme in the gel
structures
The enzyme is included in a three-dimensional network
polymer chains that form a gel.
47.
Methods of physical immobilization:3) mechanical inclusion of the enzyme in the gel
structures
Should be considered:
1. Correspondence of pore size to the size of the enzyme.
2. The nature of the matrix (since it creates
microenvironment for the enzyme, maybe
create a pH different from the pH of the solution and
increase the affinity of the substrate for the matrix, which
increases the rate of enzymatic reaction)
48.
Methods of physical immobilization:3) mechanical inclusion of the enzyme in the gel
structures
Advantages:
1) Relative simplicity of the technique
2) Increased mechanical, chemical and
thermal resistance of matrices.
3) The enzyme is stabilized
4) The enzyme is protected from bacterial
damage
Flaws:
1) Not applicable for high molecular weight
49.
Methods of physical immobilization:4) Connection to a two-phase system
The enzyme is soluble in only one of the phases, and
product - to another
Allows you to work in high molecular weight
substrates.
50.
Chemical immobilization methods:Formation of covalent bonds between
enzyme and carrier.
Advantages:
1) High strength of the conjugate
2) Enzyme stability can be increased
51.
When immobilizing enzymes, it is necessarycomply with the following conditions:
1. Active groups of the matrix should not
block the catalytic center of an enzyme.
2. Immobilization should not lead to loss
enzyme activity.
52.
Very promising is the use inas immobilized biocatalysts
cells.
Because can be avoided:
1) expensive isolation and purification steps
enzymes
2) the need for their subsequent stabilization
53.
ThermozymsStable under high temperature conditions,
high salt concentrations and extreme
pH values.
Hyperthermophilic microorganisms
found among Archaea and Bacteria, live
at temperatures of 80–100 °C.
54.
Mechanisms responsible for thermal stabilityenzymes in thermozymes:
Between mesophilic and thermophilic
enzyme versions - high degree of homology
sequences and structures.
Thus, sequences of thermostable
dehydrogenases from Pyrococcus and Thermotoga at 35 and
55% respectively identical
mesophilic dehydrogenase sequences
from Clostridium.
55.
It was discovered that dehydrogenase from Pyrococcusfuriosus (Tm == 105 °C) contains 35 isoleucines,
while dehydrogenases from Thermotoga
maritima (Tm = 95 °C) and Clostridium symbiosum (Tm
= 55 °C) only 21 and 20 isoleucines
respectively.
Heat-stable enzymes contain less
glycine: Cs dehydrogenase contains 48 residues
glycine, and dehydrogenases from Tm and Pf only
39 and 34 glycines, respectively.
More isoleucine and less glycine.
56.
Increased thermal stability correlates:1) with increasing rigidity of the protein structure
by reducing the content of residues
glycine,
2) with improvement of hydrophobic contacts in the core
dehydrogenase from Pf as a result of valine replacement
isoleucine. (As a result of site-directed
mutagenesis leading to isoleucine replacement
valine thermostability mutants
decreased).
57.
Stabilization mechanisms:minimizing the available hydrophobic area
protein surface;
optimization of protein atom packing
molecules (minimizing the ratio
surface/volume);
optimization of charge distribution (achieved
thanks to the elimination of repulsive
interactions, as well as as a result of the organization
interactions between charges into a peculiar
net)
Reducing the number of depressions
58.
Application of enzymes from extremophilesModern technologies of molecular biology
and genetic engineering allows:
1) obtain sufficient amounts of enzymes from
extremophiles for their subsequent
analysis and practical application.
2) cloning and expression of these enzymes in
mesophilic organisms.
59.
Starch is used to produce sugars.
First, the process is carried out at (95–105 °C) and at values
pH 6–6.5.
At the next stage, the temperature drops to 60°C and
pH=4.5.
The use of thermostable enzymes (αamylase, glucoamylase, xylose isomerase),
isolated from hyperthermophiles will allow:
1) carry out the process in one stage and at the same
the same conditions
2) abandon expensive ion exchangers
60.
Application of enzymes from extremophiles:The most thermostable α-amylases were
found in the archaea Pyrococcus woesei,
Pyrococcus furiosus, Desulfurococcus mucosus,
Pyrodictium abyssi and Staphylothermus
marinus. Amylase genes from Pyrococcus sp. were
cloned and expressed in E.coli and Bacillus
subtilis.
61.
Application of enzymes from extremophiles:Proteolytic enzymes
Serine alkaline proteinases are widely
used as additives to detergents
means.
Proteinases from extremophiles retain
nativeness at high temperatures, in
presence of high concentrations of detergents and
other denaturing agents. Pyrococcus,
Thermococcus, Staphylothermus, Desulfurococcus and
Sulfolobus. The maximum activity of these
enzymes develop at temperatures
from 90 to 110 °C and pH values from 2 to 10
62.
Application of enzymes from extremophiles:DNA polymerases
Thermostable DNA polymerases are used
in PCR and play an important role in genetic engineering.
Thermostable polymerases have been found in
hyperthermophiles Pyrococcus furiosus and Pyrococcus
litoralis, as well as in thermophiles Thermus aquaticus.
