The enzymatic activity it is a way of expressing the amount of the enzyme present at a given time. Indicates the amount of substrate transformed into product, by the catalytic action of the enzyme per unit of time.
It is influenced by the conditions in which the enzymatic reaction takes place, which is why it usually refers to the temperature at which it is measured. But what are enzymes? They are biological catalysts, capable of accelerating the speed of a reaction without undergoing an irreversible change during the catalyzed process..
Enzymes, in general, are proteins with the exception of ribosomes, RNA molecules with enzymatic activity.
Enzymes increase the speed of the reaction by reducing the energy barrier (activation energy); that must be expired to reach the transition state and thus the reaction occurs.
The substrate molecules that reach the transition state undergo structural changes, which lead them to originate the product molecules. Based on the functions they fulfill, enzymes are classified into six large groups: oxyreductases, transferases, hydrolases, lyases, isomerases and ligases..
The enzymes bromelain and papain, for example, are proteolytic enzymes (hydrolases) found in pineapple or pineapple, and papaya or papaya, respectively..
It is known that both pineapple and papaya facilitate the digestive process, since by acting the proteolytic enzymes they contain help to digest the proteins from, that is, meats and grains.
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The enzymatic unit (IU) is the amount of enzyme that catalyzes the transformation of 1 µmol of substrate in one minute.
Subsequently, the International System of Units (SI) defined the unit of enzyme activity as the amount of enzyme that converts 1 mole of substrate into product per second. This unit received the name of katal (kat).
1 mole = 106 µmol and 1 minute = 60 seconds.
Therefore, 1 katal equals 60106 UI. As the katal is a large unit, smaller units are often used, such as: the microkatal (µkat), 10-6 katal, and the nanokatal (πkat), 10-9 katal.
It is the number of units of enzyme activity divided by the milligrams of protein in the sample under test. The specific activity is directly related to the degree of purification of the enzyme.
There are several methods for determining the activity of an enzyme. The choice of a particular method will depend on the objective of the enzyme assay; the applicability of the method; access to the equipment necessary to conduct the experiment; the cost of using a particular method, etc..
There are spectrophotometric, fluorometric, chemiluminescence, calorimetric, radiometric and chromatographic methods..
Spectrophotometric methods can be colorimetric and read in the ultraviolet (UV) region of electromagnetic radiation..
It is based on the generation of a chromophore by enzymatic action. Enzyme activity can be followed continuously or discontinuously.
In the continuous form, the reagents are placed in a cuvette in the spectrophotometer at the desired wavelength, which corresponds to the one at which the chromophore has its maximum optical density value; and that in addition, there is no interference with another substance that may be generated.
The enzymatic reaction is initiated by the addition of the sample containing the enzyme, the activity of which is to be determined. Simultaneously, the stopwatch is started, and from time to time, the optical density value is noted.
As the equivalence of the optical density with the moles of substrate or the product of the enzymatic action is known, depending on the technique used, it is possible to calculate the moles of the substrate consumed or the moles produced..
Furthermore, since the elapsed time of the enzymatic reaction has been measured, the moles consumed or produced per second can be obtained. Thus, the enzymatic activity is established in katal units.
In the batch form for determining the enzymatic activity, the test tubes with the reaction components, except for the sample containing the enzyme or another component, are placed in a bath at 37 ° C. The reaction then starts with the addition of the missing component..
The time indicated by the technique is allowed to occur, and the reaction is terminated by the addition of a compound that stops the reaction. The optical density is read at that moment, and finally proceeds in the same way as in the continuous way to determine the enzymatic activity..
The coenzyme nicotinamityinucleotide, for example, has two forms: NADH (reduced), and NAD+ (rusty). Likewise, the coenzyme nicotinamityinucleotide phosphate has two forms NADPH and NADP+, reduced and oxidized, respectively.
Both the reduced and oxidized forms of the coenzyme are read at a length of 260 nm from ultraviolet light; meanwhile, only the reduced forms are read at a length of 340 nm from the ultraviolet light.
Therefore, both in the oxidation or reduction reactions in which the named coenzymes intervene, they are read at 340 nm.
The determination of the enzymatic activity, in essence, is the same as that followed in the continuous form of the colorimetric method; except that the optical density is read at 340 nm to observe the generation of NADH or NADPH, or to measure the consumption of these coenzymes.
This will depend on whether the measured reaction is oxidation or reduction. By means of the correspondence between the optical density and the moles of NADH and NADPH, as the case may be, the enzymatic activity can be calculated by dividing the moles of the coenzyme by the elapsed time in seconds.
As the substrate concentration increases, the enzyme activity increases. But at a certain concentration of the substrate, the active site or active sites of the enzyme are saturated, so the enzyme activity becomes constant..
However, the product of the enzymatic action can also interact with the active sites of the enzyme, producing an inhibition of the enzymatic activity..
The product can act as a competitive inhibitor; for example, the enzyme hexokinase may be mentioned. This enzyme produces the phosphorylation of glucose giving rise to glucose-6-phosphate, a compound that, when accumulated, inhibits hexokinase.
It can happen that a group of enzymes (A, B, C, D, E and F) act sequentially in a metabolic pathway. Enzyme B uses the product of Enzyme A as a substrate, and so on.
The cell, depending on its metabolic requirements, can activate or inhibit the sequences of enzymatic activities. For example, the accumulation of the product of the enzyme F, can act by inhibiting the enzyme A or any other of the enzymes of the sequence.
An enzyme can be made up of several subunits, each with its respective active sites. But these subunits do not act independently, so the activity of one of the subunits can activate or inhibit the action of the remaining ones..
Although hemoglobin is not considered an enzyme, it is a magnificent model for the phenomenon of allosterism. Hemoglobin is made up of four protein chains, two α chains and two β chains, each of them linked to a heme group..
Two phenomena can occur between the subunits: homoalosterism and heteroalosterism.
Binding of the substrate to one of the subunits increases the affinity of the other subunits for the substrate, in turn increasing the enzymatic activity of each of the remaining subunits..
Likewise, the inhibition of the enzymatic activity in one of the subunits produces the same effect in the remaining ones..
In the case of hemoglobin, the binding of oxygen to a heme group of one of the protein chains will cause an increase in the avidity for oxygen in the remaining chains..
Likewise, the release of oxygen from a heme group causes the release of oxygen from the remaining groups of the protein chains..
The binding of an activating or inhibiting substance, other than the substrate, to one of the subunits will cause an activation or inhibition of the enzymatic activity in the other subunits.
In the case of hemoglobin, the binding to the heme group of H+, COtwo and 2,3-diphosphoglycerate to one of the subunits, decreases the affinity of the heme group for oxygen, causing its release. This release of oxygen is also produced in the other chains of hemoglobin.
As the concentration of the substrate increases, so does the enzyme activity. This is due to increased access of the substrate molecules to the active sites of the enzyme..
But, for a given concentration of the substrate, all the active sites of the enzyme are saturated with this, causing that the enzymatic activity does not increase even if the concentration of the substrate is increased..
Enzymes have an optimal pH at which the affinity of the enzyme for the substrate is highest. At this pH the maximum value of enzymatic activity is reached.
The excess acidity or basicity of the medium can cause a denaturation of the enzyme, consequently reducing its activity..
The pH profile of enzyme activity is varied. Thus, for example, pepsin has a maximum activity between 1-2 pH units; trypsin has an optimum pH of 8; and papain has a constant activity between a pH range between 4 and 8.
Enzyme activity increases as temperature increases. In general, enzyme activity doubles for every 10 degrees of increase, until the optimum temperature for enzyme activity is reached..
However, when the optimum temperature is exceeded, the enzyme activity tends to decrease as the temperature of the reaction increases. This is due to the fact that proteins, and therefore enzymes, undergo denaturation due to an excessive increase in temperature..
In general, enzymes have optimal activity in a concentration range, comprised between 0 and 500 mmol / L. However, for higher concentrations, enzyme activity tends to decrease.
Under these circumstances, certain ionic interactions in enzymes, necessary for their maximum activity, are blocked..
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