Definition
Enzymes are proteins produced in plant and animal cells, which act as catalysts accelerating biological reactions without being modified.
Enzymes work by combining with a specific substance to transform it into a different substance; classic examples are given by digestive enzymes present in saliva, stomach, pancreas and small intestine, which perform an essential function in digestion and help break down food into basic constituents, which can then be absorbed and used by the body , processed by other enzymes or excreted as waste.
Each enzyme has a specific role: the one that breaks down fats, for example, does not act on proteins or carbohydrates. Enzymes are essential for the well-being of the organism. The deficiency, even of a single enzyme, can cause serious problems. A well-known example is phenylketonuria (PKU), a disease characterized by the inability to metabolize an essential amino acid, phenylalanine, the accumulation of which can cause physical deformities and mental illnesses.
Biochemical study
Enzymes are particular proteins that have the characteristic of being biological catalysts, that is, they have the ability to break down the activation energy (Eatt) of a reaction, modifying its path so that a kinetically slow process is faster.
Enzymes increase the kinetics of thermodynamically possible reactions and, unlike catalysts, they are more or less specific: they therefore possess substrate specificity.
The enzyme is not involved in the stoichiometry of the reaction: for this to happen, the final catalytic site must be identical to the starting one.
In the catalytic action there is almost always a slow phase which determines the speed of the process.
When we talk about enzymes it is not correct to speak of equilibrium reactions, we speak, instead, of steady state (state in which a certain metabolite is formed and consumed continuously, keeping its concentration almost constant over time). The product of a reaction catalyzed by one enzyme is usually itself a reactant for a subsequent reaction, catalyzed by another enzyme, and so on.
Processes catalyzed by enzymes usually consist of sequences of reactions.
A generic reaction catalyzed by an enzyme (E) can be summarized as follows:
E is the enzyme
S is the substrate;
ES represents the adduct between enzyme and substrate;
P is the product;
K is the rate constant of the reaction.
A generic enzyme (E) combines with the substrate (S) to form the adduct (ES) with a rate constant K1; it can dissociate back into E + S, with a rate constant K2, or, (if "lives" long enough) can proceed to form P with a speed constant K3.
The product (P) can, in turn, recombine with the enzyme and reform the adduct with rate constant K4.
When the enzyme and substrate are mixed, there is a fraction of time in which the meeting between the two species has not yet occurred: that is, there is an extremely short time interval (which depends on the reaction) in which enzyme and substrate have not yet met; after this period, enzyme and substrate come into contact in increasing quantities and the ES adduct is formed. Subsequently, the enzyme acts on the substrate and the product is released. It can then be said that c "is an initial time interval in which the concentration of the adduct ES is not definable; after this period, it is assumed that a steady state is established , that is, the speed of the processes which lead to obtaining the adduct is equal to the speed of the processes which lead to the destruction of the adduct.
The Michaelis-Menten constant (KM) is an equilibrium constant (referred to the first equilibrium described above); it can be said, with a good approximation (because K3 should also be considered), that KM is represented by the ratio between the kinetic constants K2 and K1 (referred to the destruction and formation of the adduct ES in the first equilibrium described above).
Through the Michaelis-Menten constant we have an "indication of the affinity between enzyme and substrate: if the KM is small c" is a "high affinity between enzyme and substrate, then the ES adduct is stable.
Enzymes are subject to regulation (or modulation).
In the past there was mainly talk of negative modulation, that is, inhibition of the catalytic capabilities of an enzyme but, there can also be a positive modulation, that is, there are species capable of enhancing the catalytic capabilities of an enzyme.
There are 4 types of inhibitions (obtained from approximations made on a model to match experimental data with mathematical equations):
- competitive inhibition
- non-competitive inhibition
- incompetitive inhibition
- acompetitive inhibition
We speak of competitive inhibition when a molecule (inhibitor) is able to compete with the substrate. For structural similarity, the inhibitor can react in place of the substrate; this is where the terminology "competitive inhibition" comes from. The probability that the enzyme binds to the inhibitor or substrate depends on the concentration of both and their affinity with the enzyme; the reaction rate therefore depends on these factors.
To obtain the same reaction rate as without the presence of the inhibitor, it is necessary to have a higher substrate concentration.
It is experimentally shown that, in the presence of an inhibitor, the Michaelis-Menten constant increases.
As regards, however, the "non-competitive inhibition, the interaction between the molecule that should function as a modulator (positive or negative-inhibitor) and the" enzyme, takes place in a site that is different from the one in which the interaction occurs. between enzyme and substrate; we therefore speak of allosteric modulation (from the Greek allosteros → other site).
If the inhibitor binds to the enzyme, it can induce a modification of the enzyme structure and, consequently, can decrease the efficiency with which the substrate binds to the enzyme.
In this type of process, the Michaelis-Menten constant remains constant since this value depends on the equilibria between the enzyme and the substrate and, even in the presence of an inhibitor, these equilibria do not change.
The phenomenon of incompetitive inhibition is rare; a typical incompetitive inhibitor is a substance that reversibly binds to the ES adduct giving rise to ESI:
Inhibition from substrate excess can sometimes be incompetitive, as this occurs when a second substrate molecule binds to the ES complex, giving rise to the ESS complex.
An acompetitive inhibitor, on the other hand, can only bind to the substrate enzyme adduct as in the previous case: the binding of the substrate to the free enzyme induces a conformational modification which makes the site accessible for the inhibitor.
The Michaelis Menten constant decreases as the inhibitor concentration increases: apparently, therefore, the affinity of the enzyme for the substrate increases.
Serine protease
They are a family of enzymes to which chymotrypsin and trypsin belong.
Chymotrypsin is a proteolytic and hydrolytic enzyme that cuts to the right of hydrophobic and aromatic amino acids.
The product of the gene that codes for chymotrypsin is not active (it is activated with a command); the inactive form of chymotrypsin is represented by a polypeptide chain of 245 amino acids. Chymotrypsin has a globular shape due to five disulfide bridges and other minor interactions (electrostatic, Van der Waals forces, hydrogen bonds, etc.).
Chymotrypsin is produced by the chymose cells of the pancreas where it is contained in special membranes and expelled through the pancreatic duct into the intestine, at the time of digestion of food: chymotrypsin is in fact a digestive enzyme. The proteins and nutrients that we ingest through the diet are subjected to digestion to be reduced to smaller chains and to be absorbed and transformed into energy (e.g. amylase and protease break down nutrients into glucose and amino acids that reach the cells, through the blood vessels they reach the portal vein and from there are conveyed to the liver where they undergo further treatment).
Enzymes are produced in a non-active form and are activated only when they reach the "site where they must operate"; once their action is finished, they are deactivated. An enzyme, once deactivated, cannot be reactivated: to have a "further catalytic action, it must be replaced by" another enzyme molecule. If chimitrypsin were produced in active form already in the pancreas, it would attack the latter: pancreatitis are pathologies due to digestive enzymes that are already activated in the pancreas (and not in the required sites); some of them if not treated in time, lead to death.
In chymotrypsin and in all serine proteases, the catalytic action is due to the existence of the alcohol anion (-CH2O-) in the side chain of a serine.
Serine proteases take this name precisely because their catalytic action is due to a serine.
Once all the enzyme has performed its action, before being able to re-operate on the substrate again, it must be restored with water; the "release" of serine by the water is the slowest stage of the process, and it is this phase which determines the speed of catalysis.
The catalytic action occurs in two phases:
- formation of the anion with catalytic properties (anion alcoholate) and subsequent nucleophilic attack on the carbonyl carbon (C = O) with cleavage of the peptide bond and formation of the ester;
- water attack with restoration of the catalyst (able to exert its catalytic action again).
The various enzymes belonging to the serine protease family can be made up of different amino acids but, for all of them, the catalytic site is represented by the alcoholic anion of the side chain of a serine.
A subfamily of serine proteases is that of the enzymes involved in coagulation (which consists in the transformation of protein, from their inactive form to an "other form that is active). These enzymes ensure that coagulation is as effective as possible and is limited in the space and time (coagulation must occur quickly and must occur only in the vicinity of the injured area). The enzymes involved in coagulation are activated in a cascade (from the activation of a single enzyme, billions of enzymes are obtained: each activated enzyme, in turn activates many other enzymes).
Thrombosis is a pathology due to the malfunctioning of coagulation enzymes: it is caused by the activation, without necessity (because there is no injury), of the enzymes used in coagulation.
There are modulatory (regulatory) enzymes and inhibitory enzymes for other enzymes: interacting with the latter, they regulate or inhibit their activity; even the product of an enzyme can be an inhibitor for the enzyme. There are also enzymes that work the more, the greater the substrate present.
Lysozyme
Luigi Pasteur discovered, by sneezing on a petri dish, that in the mucus there is an enzyme capable of killing bacteria: lysozyme; from the Greek: liso = what size; zimo = enzyme.
Lysozyme is capable of breaking down the cell wall of bacteria. Bacteria, and unicellular organisms in general, need mechanically resistant structures that limit their shape; inside the bacteria there is a very high osmotic pressure so they attract water. The plasma membrane would explode if there was no cell wall that opposes the entry of water and limits the volume of the bacterium.
The cell wall consists of a polysaccharide chain in which molecules of N-acetyl-glucosamine (NAG) and molecules of N-acetyl-muramic acid (NAM) alternate; the bond between NAG and NAM is broken by hydrolysis. The NAM carboxyl group in the cell wall is engaged in a peptide bond with an amino acid.
Between the various chains, bridges are formed consisting of pseudo-peptide bonds: the branching is due to the lysine molecule; the structure as a whole is very branched and this gives it a high stability.
Lysozyme is an antibiotic (kills bacteria): it works by making a crack in the bacterial wall; when this structure (which is mechanically resistant) breaks, the bacterium draws water until it bursts. Lysozyme manages to break the β-1,4 glucosidic bond between NAM and NAG.
The catalytic site of lysozyme is represented by a groove that runs along the enzyme in which the polysaccharide chain is inserted: six glucosidic rings of the chain are placed in the groove.
In position three of the groove c "is a choke: in this position only one NAG can be placed, because the NAM, which is of higher dimensions, cannot enter. The actual catalytic site is between positions four and five: since there is a NAG in position three, the cut will take place between a NAM and a NAG (and not vice versa); the cut, therefore, is specific.
The optimal pH for lysozyme to work is five. In the catalytic site of the enzyme, ie between positions four and five, there are the side chains of an aspartic acid and a glutamic acid.
Degree of homology: measures the kinship (i.e. similarity) between protein structures.
There is a strong relationship between lysozyme and lactose-synthase.
Lactose synthetase synthesizes lactose (which is the main milk sugar): lactose is a galactosyl glucoside in which c "is a β-1,4 glucosidic bond between galactose and glucose.
Therefore, lactose synthetase catalyzes the opposite reaction to that catalyzed by lysozyme (which instead splits the β-1,4 glucosidic bond)
Lactose synthetase is a dimer, that is, it is made up of two protein chains, one of which has catalytic properties and is comparable to lysozyme and the other is a regulatory subunit.
During pregnancy, glycoproteins are synthesized by the cells of the mammary gland by the action of galatosyl-tranferase (it has a "sequence homology of 40% with lysozyme): this enzyme is able to transfer a galactosyl group from a high-energy structure to a glycoprotein structure. During pregnancy, the expression of the gene that codes for galactosisyl-transferase is induced (there is also the expression of other genes that also give other products): there is an increase in the size of the breast because it is activated the mammary gland (previously inactive) which must produce milk. During childbirth, α-lactalbumin is produced which is a regulatory protein: it is able to regulate the catalytic capacity of galactosyl-transferase (by discrimination of the substrate) . Galactosyl-transferase modified by α-lactalalbumin is able to transfer a galactosyl onto a glucose molecule: forming a β-1,4 glycosidic bond and giving lactose (lactose synthetase).
Hence, galactose transferase prepares the mammary gland before delivery and produces milk after delivery.
To produce glycoproteins, galactosyl transferase binds to a galactosyl and a NAG; during childbirth lactal albumin binds to galactosyltransferase making the latter recognize glucose and no longer NAG to give lactose.
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