Why can reversible reactions take place
Enzyme inhibition is the adverse effect on an enzymatic reaction by an inhibitor called an inhibitor. The speed of the reaction is thereby reduced. The inhibitors can bind to different substances involved in the reaction, such as the enzyme or the substrate. The binding site on the enzyme can also vary from the active site at which the substrate binds to other sites that are important for the activity of the enzyme.
Enzymes are essential for every organism. They are involved in every metabolic process and act as catalysts for most reactions. In order to be able to regulate these processes, the cells need certain mechanisms that influence the activity of the enzymes. Some enzymes can be switched on by modifications, i.e. activated. For example, the pyruvate kinase required for the utilization of glucose is regulated by phosphorylation, i. H. a phosphoryl group can be attached to the enzyme. This phosphorylated form of pyruvate kinase is not very active. However, if the enzyme has not been modified by a phosphoryl group, it is fully active.
The activity of enzymes can also be influenced by the binding of certain substances. These substances are called effectors. Depending on how effectors act on an enzyme, they are called activators or inhibitors. Activators increase the activity of enzymes, i. H. they promote the reaction catalyzed by the enzyme. Inhibitors have a negative effect on the enzyme. They lower the activity and thus inhibit the reaction catalyzed by the enzyme. This is called enzyme inhibition.
There are other ways to reduce enzyme activity, but they are not enzyme inhibition. These include influences from temperature, pH, ionic strength or solvent effects. These factors act unspecifically, for example by changing the conformation of the enzyme, i.e. the spatial structure, without specifically affecting the active center of the enzyme. The active center is, so to speak, the place on the enzyme where the substrate binds, i.e. the reaction also takes place.
Classification of enzyme inhibition
The enzyme inhibition is divided into reversible and irreversible inhibition, depending on the binding of the inhibitor.
In the case of reversible enzyme inhibition, the inhibitor can be split off or displaced by the enzyme again. It does not bind tightly to the enzyme. This form of enzyme inhibition is used to regulate various metabolic processes that should not take place at times. For example, glycolysis is used to generate energy from glucose. One of these enzymes has already been mentioned above, the pyruvate kinase. Another glycolysis enzyme is phosphofructokinase. If there is a lot of energy in the cell, it has this stored in the form of adenosine triphosphate (ATP). As an inhibitor, this ATP inhibits both phosphofructokinase and pyruvate kinase. This means that glucose is no longer converted into energy, i.e. ATP. This special form of enzyme inhibition, in which the end product inhibits the enzyme that leads to the synthesis of this substance, is called end product inhibition or feed-back inhibition.
In the case of irreversible inhibition, the inhibitor binds so tightly that it can no longer be detached from the enzyme. The activity of the enzyme is lost. The irreversible inhibition can be found, for example, in fungi that produce antibiotics to protect them. These antibiotics often irreversibly inhibit certain metabolic pathways such as protein biosynthesis.
Reversible enzyme inhibition
In the case of reversible enzyme inhibition, the inhibitor I binds reversibly to the enzyme E and thus lowers its activity or the speed of the reaction of the substrate S to the product P. However, the inhibitor can, for example, be displaced by the substrate again. From a mathematical point of view, there is the rate constantk2 the "speed" of the uninhibited reaction. If the inhibitor now binds to the enzyme-substrate complex ES, the reaction is controlled by the rate constant k6 Are defined. k6 is smaller than k2. Since it is a reversible enzyme inhibition, an equilibrium is established in the reactions to ES, the enzyme-inhibitor complex EI and the enzyme-substrate-inhibitor complex ESI. All rate constants also for the equilibria are shown in Fig. 1 by the corresponding reaction arrows.
In steady state, the reaction rate can even be determined mathematically:
By reformulating the following equation for the reaction rate, which is generally valid for reversible enzyme inhibition:
The constant introduced V.1 corresponds to k2[E.0] and the constant V.2 = k6[E.0]. The equilibrium constants Kic and Kiu can be derived from Fig. 1: Kic = k − 3 / k3 and Kiu = k − 4 / k4.
The most important mechanisms of the reversible enzyme inhibition are shown in Fig. 2. The division was made into complete and partial inhibition, which are divided into V.2 distinguish. When the enzyme is completely inhibited V.2 at 0, for the partial this value is not equal to 0. This means that the enzyme retains its catalytic activity in the case of a partial inhibition, although this is influenced by the inhibitor. In the case of complete enzyme inhibition, however, the ESI complex can no longer participate in the reaction and is therefore inactive.
Product inhibition is a special case of competitive inhibition because the inhibitor corresponds to the product of the reaction. The substrate excess inhibition is also a special case of the uncompetitive inhibition. In this case, the substrate is the inhibitor if it is present in high concentration.
Competitive inhibitors are substances that compete with the substrate for the binding site in the active center of the enzyme. They are not converted and can therefore be displaced by the substrate again. Competitive inhibitors are often very similar to the substrate. The mechanism of competitive inhibition is shown in Figure 3. It can be clearly seen that the enzyme E cannot bind the substrate S and the inhibitor I at the same time. The reversible binding of S or I to E creates an equilibrium between free enzyme E, the enzyme-substrate complex ES and the enzyme-inhibitor complex EI. Provided that both the substrate S and the inhibitor I are present in much higher concentrations than the enzyme E, the following reaction rate equation can be formulated for the steady-state state:
The constants contained are defined as follows:
The reaction speed depends on the starting substrate concentration (S.0) in the absence and presence of the competitive inhibitor shown in Fig. 4. The Michaelis-Menten constant is increased by the factor i in the presence of the inhibitor. The maximum speed V.max however remains unchanged.
The linearization of the Michaelis-Menten plot is achieved by the reciprocal of the equation.
The double reciprocal application, i.e. the application of 1 / v against 1 / [S.0] in the presence of different concentrations of the inhibitor is shown in Fig. 5 and is called the Lineweaver-Burk plot. The slope of the graph increases by i in the presence of the inhibitor. There V.max remains unchanged, all straight lines intersect on the ordinate at point 1 / V.max. The points of intersection with the abscissa reflect the value - 1 / Kmi contrary.
The equations described above were derived for competitive inhibition in which the simultaneous binding of substrate and inhibitor is excluded. However, the conditions for competitive inhibition can also be met if the inhibitor does not occupy the same binding site on the enzyme as the substrate. Binding in the active center, which sterically restricts substrate binding, also leads to the competitive inhibitor type.
Inhibition by a competing substrate
In this special case of reversible enzyme inhibition, the enzyme is able to catalyze two reactions, i.e. to bind two different substrates. The enzyme E binds the substrate A and converts it to the product P. The binding of the substrate B to the enzyme E leads to the formation of product Q. Thus, the reaction rate vA. reduced by adding the substrate B, since the second substrate B can also be bound and converted. In this competitive reaction, one substrate has an effect on the reaction rate of the reaction of the respective
other substrate as a competitive inhibitor. This results in the following
In the case of non-competitive inhibition, the binding of the inhibitor I to the enzyme E does not affect the substrate binding. The inhibitor I is thus able to bind both to the free enzyme E and to the enzyme-substrate complex ES, i. H. the inhibitor does not bind in the substrate-binding part of the enzyme, the active site. The substrate can also react with the enzyme-inhibitor complex EI, but the enzyme-inhibitor-substrate complex EIS that is formed is not able to split off the product P. The reaction mechanism is shown in more detail in Fig. 6.
A simple rate equation can be derived under steady-state conditions:
The constants Km and i are defined as follows: Km = k − 1 / k1 and i = 1 + (k3[I.0] / k − 3). From the rate equation one can deduce that the non-competitive inhibitor in the presence of the maximum rate V.max by a factor of 1 / i decreased. The Km-Value for the substrate remains unchanged.
The application according to Lineweaver-Burk takes place with the formation of the reciprocal reaction rate according to this formula:
The Lineweaver-Burk plot is shown in Fig. 8. The increase after the addition of the non-competitive inhibitor is i-fold higher than without inhibition. The ordinate intersection of each straight line is included i / V.max. The straight lines for reactions of different inhibitor concentrations intersect at exactly one point on the abscissa, in the value - 1 / Km.
In some cases of non-competitive inhibition, the behavior of the inhibitor deviates somewhat from the "normal case". The reaction of the inhibitor with the enzyme then takes place much faster than that of the substrate. At low substrate concentrations, the reduction in maximum speed is not so great. Hence it results for the conditions k − 1 <><>k2 and Km = k2 / k1 following speed equation:
Such behavior should be expected when determining the type of inhibitor of an inhibitor.
Occasionally, in addition to competitive and non-competitive inhibition, there is also the uncompetitive type of inhibition. The inhibitor only reacts with the enzyme-substrate complex ES, as can be seen in Fig. 9. The equation for the reaction rate derived for this mechanism under steady-state conditions is as follows:
The constants are defined as follows: i = 1 + (k3[I.0] / k − 3 and Km = (k − 1 + k2) / k1. From the Michaelis-Menten plot of an uncompetitive inhibition shown in Fig. 10, it can be seen that the inhibitor increases both the maximum reaction rate and the KmValue is changed.
By transforming the rate equation into the reciprocal form, the dependence of the reaction rate on the initial concentration of the substrate can be represented in a linearized manner:
From this formula it can be seen that the increase is independent of the uncompetitive inhibitor, i.e. the graphs are parallel to one another for different inhibitor concentrations. The zero gives the value i / Km again. The straight lines intersect the ordinate at the point i / V.max.
The uncompetitive inhibition occurs, for example, with oxidases when the inhibitor can only react with a certain oxidation level of the enzyme. Another possibility for an uncompetitive inhibitor is offered by an ordered mechanism, a two-substrate reaction in which the inhibitor competes with one of the substrates.
Partly competitive inhibition
One speaks of a partially competitive inhibition when the inhibitor binding (I) only reduces the affinity of the enzyme E for the substrate S without influencing the rate constant for product formation (P). This means that even after the inhibitor has been bound, product can be formed. Fig. 11 shows the mechanism of partially competitive inhibition.
For one under these conditions:
- rapid equilibrium (binding of the substrate or inhibitor much faster than formation of the product)
- high inhibitor concentrations
If the type of inhibition is partially competitive, the reaction rate can be calculated as follows:
From this velocity equation it can be seen that the Ks- Values itself against the competitive inhibition of the value K's approximates. In the Lineweaver-Burk diagram, the partially competitive inhibition cannot be distinguished from the competitive inhibition. In order to differentiate these, however, one uses the dependence of the Ks-Value of the inhibitor concentration shown in Fig. 12. In the case of competitive inhibition, this dependency is represented by a straight line in the diagram. As can be seen in the figure, this situation does not apply to partially competitive inhibition.
Inhibition of excess substrate
With some enzymes, very high substrate concentrations can bind a second substrate molecule to the enzyme. The resulting ESS complex is unable to break down into product and enzyme. The rate equation for this reaction mechanism is as follows:
The dissociation constant of the ESS complex was taken as Ki designated. If the KmValue much lower than that KiValue, a hyperbolic dependence is obtained in the Michaelis-Menten plot (Fig. 13, curve 1). Are these two values approximately the same or the KmIf the value is higher, an optimum curve is created, so to speak (Fig. 13, curve 2).
Inhibition by reaction of an inhibitor with the substrate
With this type of inhibitor, the inhibitor reacts with the substrate, which is then no longer converted by the enzyme. The binding of the inhibitor is regarded as reversible in this inhibition. As a result, the substrate concentration freely accessible to the enzyme [S.eff] reduced:
In the presence of the inhibitor, the maximum speed is reached at high substrate concentrations. In the Lineweaver-Burk diagram, this mechanism can be differentiated from a competitive inhibitor, since a deviation from linearity occurs.
Irreversible enzyme inhibition
The irreversible binding of the inhibitor to the enzyme reduces the catalytic activity. A dissociation of the enzyme-inhibitor complex into free enzyme and inhibitor is not possible; H. the enzyme remains inactive forever. The activity depends linearly on the inhibitor concentration. This dependency can be seen in Fig. 14 (curve 1). Deviating from this curve, curve 2 arises in the figure. The inhibitor reacts irreversibly with several groups of different specificities, which leads to titration of the more specific groups that are not actively involved in the catalytic mechanism. The groups that are important for catalysis then react, which reduces the activity. On the other hand, the inhibitor can enter into a complex with the enzyme which still has little activity. This is the case with curve 3 of the figure.
If the inhibitor concentration is considerably higher than that of the enzyme, the rate constant for the reaction of the enzyme with the inhibitor can be formulated as a pseudo-first order reaction:
A corresponds to the activity in this equation. The rate constant k 'can be determined from the increase by plotting the logarithm of the activity against the reaction time.
The rate constant is influenced by the presence of the substrate. This is because this protects the enzyme from inactivation by the inhibitor, which means that the rate constant is lower than without the addition of substrate. With such behavior, the inhibitor may bind to a specific group in the active center, i.e. it may have the same binding site as the substrate.
An example of irreversible inhibition are the so-called “suicide substrates”, which enter into a covalent bond with the functional group of the enzyme and thus block it.
Enzyme kinetics, Michaelis-Menten theory, catalytic efficiency
- Hans Bisswanger: Enzyme kinetics. Theory and methods Wiley-VCH, 2000, ISBN 3-52-730096-1
- Alfred Schellenberger (Ed.): Enzyme Catalysis: Introduction to the chemistry, biochemistry, and technology of enzymes Gustav Fischer Verlag, Jena 1989
Category: Biochemical Reaction
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