The active site of the enzyme does not. Active center of enzymes

Study of the mechanism of a chemical reaction catalyzed by an enzyme along with the determination of intermediate and final products on different stages reaction implies accurate knowledge of the geometry of the tertiary structure of the enzyme, the nature of the functional groups of its molecule, providing specificity of action and high catalytic activity on a given substrate, as well as the chemical nature of the site (s) of the enzyme molecule, which provides a high rate of catalytic reaction. Typically, the substrate molecules involved in enzymatic reactions are relatively small compared to enzyme molecules. Thus, during the formation of enzyme-substrate complexes, only limited fragments of the amino acid sequence of the polypeptide chain enter into direct chemical interaction - the "active center" - a unique combination of amino acid residues in the enzyme molecule, providing direct interaction with the substrate molecule and direct participation in the act of catalysis

In the active center, conditionally allocate

catalytic center - directly chemically interacting with the substrate;

binding center (contact or "anchor" site) - providing a specific affinity for the substrate and the formation of an enzyme-substrate complex.

To catalyze a reaction, an enzyme must bind to one or more substrates. The protein chain of the enzyme folds in such a way that a gap or cavity is formed on the surface of the globule, where substrates bind. This area is called the substrate binding site. Usually it coincides with the active center of the enzyme or is located near it. Some enzymes also contain binding sites for cofactors or metal ions.

The enzyme, connecting with the substrate:

cleans the substrate from the water "coat"

places the reacting substrate molecules in space in the manner necessary for the reaction to proceed

prepares for the reaction (for example, polarizes) substrate molecules.

Usually, the attachment of the enzyme to the substrate occurs due to ionic or hydrogen bonds, rarely due to covalent bonds. At the end of the reaction, its product (or products) are separated from the enzyme.

As a result, the enzyme reduces the activation energy of the reaction. This is because in the presence of an enzyme, the reaction follows a different path (in fact, a different reaction occurs), for example:

In the absence of an enzyme:

In the presence of an enzyme:

• AF + B = AVF

  AVF = AV + F

where A, B are substrates, AB is a reaction product, F is an enzyme.

Enzymes cannot independently provide energy to endergonic reactions (for which energy is required). Therefore, enzymes that carry out such reactions combine them with exergonic reactions that release more energy. For example, reactions for the synthesis of biopolymers are often coupled with the reaction of ATP hydrolysis.

The active centers of some enzymes are characterized by the phenomenon of cooperativity.

Specificity

Enzymes usually exhibit high specificity for their substrates (substrate specificity). This is achieved by partial complementarity of the shape, charge distribution, and hydrophobic regions on the substrate molecule and at the substrate binding site on the enzyme. Enzymes usually also demonstrate a high level of stereospecificity (they form only one of the possible stereoisomers as a product or only one stereoisomer is used as a substrate), regioselectivity (they form or break a chemical bond in only one of the possible positions of the substrate) and chemoselectivity (they catalyze only one chemical reaction of several possible for the given conditions). Despite the overall high level of specificity, the degree of substrate and reaction specificity of enzymes can be different. For example, trypsin endopeptidase breaks the peptide bond only after arginine or lysine, if not followed by proline, and pepsin is much less specific and can break the peptide bond following many amino acids.

8.7.1. In the cellular content, enzymes are not distributed chaotically, but in a strictly ordered manner. With the help of intracellular membranes, the cell is divided into compartments or compartments(Figure 8.18). In each of them strictly defined biochemical processes and the corresponding enzymes or polyenzyme complexes are concentrated. Here are some typical examples.

Figure 8.18. Intracellular distribution of enzymes of various metabolic pathways.

Lysosomes contain mainly various hydrolytic enzymes. Here, the processes of decomposition of complex organic compounds into their structural components take place.

The mitochondria contain complex systems redox enzymes.

Enzymes for activating amino acids are distributed in the hyaloplasm, but they are also present in the nucleus. In the hyaloplasm, numerous glycolysis metabolones are present, structurally combined with those of the pentose phosphate cycle, which provides the relationship between the dichotomous and apotomic pathways of carbohydrate breakdown.

At the same time, enzymes that accelerate the transfer of amino acid residues to the growing end of the polypeptide chain and catalyze some other reactions in the process of protein biosynthesis are concentrated in the ribosomal apparatus of the cell.

In the cell nucleus, nucleotidyl transferases are mainly localized, which accelerate the reaction of transfer of nucleotide residues during the formation of nucleic acids.

8.7.2. The distribution of enzymes in subcellular organelles is studied after preliminary fractionation of cell homogenates by high-speed centrifugation, determining the content of enzymes in each fraction.

The localization of this enzyme in a tissue or cell can often be established in situ by histochemical methods ("histoenzymology"). For this, thin (from 2 to 10 μm) sections of frozen tissue are treated with a substrate solution to which this enzyme is specific. In those places where the enzyme is located, a product of the reaction catalyzed by this enzyme is formed. If the product is colored and insoluble, it remains at the site of formation and allows the localization of the enzyme. Histoenzymology gives a clear and, to a certain extent, physiological picture of the distribution of enzymes.

Enzyme systems of enzymes, concentrated in intracellular structures, are finely coordinated with each other. The interrelation of the reactions catalyzed by them ensures the vital activity of cells, organs, tissues and the organism as a whole.

When studying the activity of various enzymes in tissues healthy body you can get a picture of their distribution. It turns out that some enzymes are widely distributed in many tissues, but in different concentrations, while others are very active in extracts obtained from one or more tissues, and are practically absent in other tissues of the body.

Figure 8.19. The relative activity of some enzymes in human tissues, expressed as a percentage of the activity in the tissue with the maximum concentration of this enzyme (Moss, Butterworth, 1978).

8.7.3. The concept of enzymopathies. In 1908, the English physician Archibald Garrod suggested that the cause of a number of diseases may be the absence of any of the key enzymes involved in metabolism. He introduced the concept of "inborn errors of metabolism" (congenital metabolic defect). Later, this theory was confirmed by new data obtained in the field of molecular biology and pathological biochemistry.

Information about the amino acid sequence in the polypeptide chain of a protein is recorded in the corresponding region of the DNA molecule in the form of a sequence of trinucleotide fragments - triplets or codons. Each triplet encodes a specific amino acid. This correspondence is called the genetic code. Moreover, some amino acids can be encoded using several codons. There are also special codons that are signals to start and stop the synthesis of the polypeptide chain. By now genetic code completely decrypted. It is universal for all types of living organisms.

The implementation of the information embedded in the DNA molecule includes several stages. First, messenger RNA (mRNA), which enters the cytoplasm, is synthesized in the cell nucleus during transcription. In turn, mRNA serves as a matrix for translation - the synthesis of polypeptide chains on ribosomes. Thus, the nature of molecular diseases is determined by the violation of the structure and function of nucleic acids and proteins controlled by them.

8.7.4. Since information about the structure of all proteins in a cell is contained in the DNA nucleotide sequence, and each amino acid is defined by a triplet of nucleotides, a change in the primary structure of DNA can ultimately have a profound effect on the synthesized protein. Such changes occur due to DNA replication errors, when one nitrogenous base is replaced by another, either as a result of radiation or chemical modification. All inherited defects arising in this way are called mutations... They can lead to incorrect reading of the code and deletion (loss) of a key amino acid, substitution of one amino acid for another, premature stop of protein synthesis, or the addition of amino acid sequences. Taking into account the dependence of the spatial packing of a protein on the linear sequence of amino acids in it, it can be assumed that such defects can change the structure of the protein, and hence its function. However, many mutations are only found in vitro and do not adversely affect protein function. Thus, key point is the localization of changes in the primary structure. If the position of the substituted amino acid turns out to be critical for the formation of the tertiary structure and the formation of the catalytic center of the enzyme, then the mutation is serious and can manifest itself as a disease.

The consequences of a deficiency of one enzyme in the chain of metabolic reactions can manifest themselves in different ways. Suppose that the transformation of the compound A in connection B catalyzes enzyme E and what's the connection C occurs on an alternative path of transformations (Figure 8.20):

Figure 8.20. Scheme of alternative pathways of biochemical transformations.

The consequences of enzyme deficiency can be the following phenomena:

1. insufficiency of the product of the enzymatic reaction ( B). As an example, we can point to a decrease in blood glucose in some forms of glycogenosis;
2. accumulation of matter ( A), the conversion of which is catalyzed by an enzyme (for example, homogentisic acid in alkaptonuria). In many lysosomal storage diseases, substances that normally undergo hydrolysis in lysosomes accumulate in them due to a deficiency of one of the enzymes;
3. deviation to an alternative path with the formation of some biologically active connections (C). This group of phenomena includes the urinary excretion of phenylpyruvic and phenyl lactic acids formed in the body of patients with phenylketonuria as a result of the activation of auxiliary pathways for the breakdown of phenylalanine.

If metabolic conversion as a whole is regulated according to the principle of feedback by the final product, then the effects of the last two types of anomalies will be more significant. So, for example, with porphyrias (congenital disorders of heme synthesis), the suppressive effect of heme on the initial synthesis reactions is eliminated, which leads to the formation of excess amounts of intermediate products of the metabolic pathway, which have toxic effect on the cells of the skin and nervous system.

Factors external environment can enhance or even completely determine clinical manifestations some congenital metabolic disorders. For example, in many patients with glucose-6-phosphate dehydrogenase deficiency, the disease begins only after taking drugs such as primaquine. In the absence of contact with medicines such people appear to be healthy.

8.7.5. Enzyme deficiency is usually judged indirectly by an increase in the concentration of the starting substance, which normally undergoes transformations under the action of this enzyme (for example, phenylalanine in phenylketonuria). Direct determination of the activity of such enzymes is carried out only in specialized centers, but if possible, the diagnosis should be confirmed by this method. Prenatal (prenatal) diagnosis of some congenital metabolic disorders is possible by examining amniotic fluid cells obtained on early stages pregnancy and cultured in vitro.

Some congenital metabolic disorders are treatable by delivering the missing metabolite to the body or by limiting the intake of gastrointestinal tract precursors of disturbed metabolic processes. Occasionally, build-up products (such as iron in hemochromatosis) can be removed.

1. Active center Is relatively small plot, located in a narrow hydrophobic depression (gap) on the surface of the enzyme molecule, directly involved in catalysis.

2. Active centers of enzymes are formed at the level of the tertiary structure.

3. Enzymatic catalysis requires precise spatial organization of large ensembles built from amino acid residues and their side groups. Such ensembles form both active and regulatory (allosteric) centers of enzymes.

4. Active center, except catalytic section, includes substrate-binding a site that is responsible for the specific complementary binding of the substrate and the formation of an enzyme-substrate complex (ES); the active site of an enzyme often includes a cofactor binding site or domain.

Example 1. Active centers of enzymes are formed at the level of the tertiary structure.

Serine proteases are widespread in nature and, together with proteolytic enzymes of other classes (aspartyl, cysteine, and metalloproteinases), provide protein cleavage (catabolism) and a number of limited proteolysis reactions that have regulatory significance for cell life.

V In different serine proteases, these amino acids can occupy different places in the peptide chain of the enzyme, but they approach each other during the folding of the polypeptide chain and their relative position in space is strictly preserved (Fig. 2.3).

5. The active center cannot be delineated by strictly defined boundaries, since each of its components interacts in one way or another with other parts of the enzyme molecule. The influence of the microenvironment can be very significant: - the components of the active center, including co-factors, interact with neighboring groups of the enzyme, which modifies the chemical characteristics of functional groups, participating in catalysis;

6. The structure of the active center determines the specificity of the enzyme action. Most enzymes are highly specific to both nature and the pathway of substrate transformation.

7. Specificity to the substrate is due to the complementarity of the structure of the substrate-binding center of the enzyme to the structure of the substrate (Fig. 2.4).

As Fig. 2.4, substrate binding site in shape corresponds to the substrate (geometric correspondence); moreover, specific bonds (hydrophobic, ionic and hydrogen) are formed between the amino acid residues of the active center of the enzyme and the substrate, i.e. installed electronic or chemical correspondence.