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Competitive Inhibition - v vs S - Biology

Competitive Inhibition - v vs S - Biology



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Competitive Inhibition - v vs S

Competitive inhibition

Competitive inhibition is interruption of a chemical pathway owing to one chemical substance inhibiting the effect of another by competing with it for binding or bonding. Any metabolic or chemical messenger system can potentially be affected by this principle, but several classes of competitive inhibition are especially important in biochemistry and medicine, including the competitive form of enzyme inhibition, the competitive form of receptor antagonism, the competitive form of antimetabolite activity, and the competitive form of poisoning (which can include any of the aforementioned types).


Reversible Enzyme Inhibition | Microbiology

The main feature of competitive inhibition is that it can be reversed by increasing the substrate concentration in a reaction mixture which contains both the substrate and the inhibitor. The degree of inhibition depends on the relative concentrations of the substrate and the inhibitor. If the substrate concentration is increased, keeping the inhibitor concentration fixed, the degree of inhibition of the enzyme activity decreases.

An opposite effect is observed, if the inhibitor concentration is increased, keeping the substrate concentration fixed. This happens because the substrate and the inhibitor both bind to the same catalytic site of the enzyme by virtue of a similarity in structure of the substrate and the inhibitor. Thus, the substrate and the inhibitor compete with each other for occupying the same active site or sites of an enzyme molecule.

Because of structural similarity, the enzyme molecule cannot distinguish between the correct substrate and the false one which is the inhibitor. An often cited example of competitive inhibition is the inhibition of succinic acid dehydrogenase by malonic acid. In the TCA cycle succinic acid is dehydrogenated by succinic dehydrogenase to fumaric acid, FAD acting as H-acceptor. In presence of malonic acid, the enzyme can combine with the inhibitor, but fails to dehydrogenate it.

The structures of succinic and malonic acids are shown:

Another well-known example of competitive inhibition having clinical importance is that of sulfanilamide and p-amino benzoic acid. Sulfanilamide forms the nucleus of all sulfa-drugs which are used as chemotherapeutic agents against a variety of infections caused by bacteria. Par-amino-benzoic acid (p-ABA) is an essential vitamin required by many bacteria for synthesis of folic acid which acts as a coenzyme.

The enzyme which acts on p-ABA to convert it to the next intermediate in the biosynthesis of folic acid is competitively inhibited by sulfanilamide, because p-ABA has structural similarity with the inhibitor. The bacteria are deprived of folic acid and are unable to grow.

The structures of the two compounds are shown below:

Non-Competitive Inhibition:

A non-competitive inhibition cannot be reversed by increasing substrate concentration, because the inhibitor does not bind to the enzyme protein at the same active site as the normal substrate, but at a different site. Hence there is no competition between the substrate and the inhibitor.

The inhibition may be caused due to a change in the shape of the substrate-site due to binding of the inhibitor to the same enzyme molecule though at a different site. This type of non-competitive inhibition is also known as allosteric inhibition and has been dealt with separately.

The more common type of non-competitive inhibition is that exerted by the heavy metal ions that bind reversibly with the sulfhydryl group (-SH) of cysteine residues of enzyme proteins. For many enzymes, the free -SH groups are essential for catalytic activity, because they are often involved in maintaining the correct three-dimensional configuration of the enzyme protein required for its catalytic function. Heavy metal ions, like Hg ++ and Ag ++ bind to the -SH groups of enzyme proteins reversibly, causing non-competitive inhibition.

Non-competitive inhibition may also be due to some agents which bind inorganic co-factors required by certain apo-enzymes to form functional holoenzymes. These inorganic co-factors are usually divalent metal ions, like Mg ++ , Ca ++ , Fe ++ etc. The inhibitors which bind such metal ions include cyanide which binds Fe ++ or Fe +++ , fluoride which binds Ca ++ or Mg ++ , ethylene diamino tetra acetic acid (EDTA) which binds Mg ++ and other divalent metal ions, etc.

Whether an inhibior acts as a competitive or a non-competitive one can be recognized from their kinetics. Lineweaver-Burk plots using varying concentrations of the inhibitor and a fixed concentration of the substrate reveal the difference between a competitive and a non-competitive inhibitor. In case of competitive inhibition, plots of 1/v against 1/[S] produce straight lines of different slopes, intersecting the 1/v axis at a common point.

The curves indicate that Vmax is not altered by the presence of the inhibitor, but the Km is. In presence of the inhibitor Km has a higher value (Fig. 8.39 A). In case of non­competitive inhibition, on the other hand, the straight lines also show different slopes with varying concentrations of the inhibitor, but they do not intercept the 1/v axis at the same point as observed in case of competitive inhibition.

Rather the lines meet at a common point on the -1/[S] axis. This indicates that increasing concentration of inhibitor causes decrease in Vmax which is not restored by increasing substrate concentration. The Km, however, remains unchanged, because the substrate and the inhibitor do not bind to the same site of the enzyme (Fig. 8.39B).


Regulation of Enzyme Activity by Activation or Inhibition | Biochemistry

In connection with the tryptophan operon, that an excess of tryptophan can cause a repression of the genes of this operon, leading to an arrest of the synthesis of the enzymes required for the formation of tryptophan.

Beside this possibility of repression, there is very often feedback inhibition, i.e. the possibility for an essential metabolite (amino acid, nucleotide, etc.), which is the final product of a series of biosynthetic reactions, of inhibiting the activity of an enzyme catalyzing one of the first reactions of this series.

The inhibited enzyme is generally the one which catalyzes the first reaction leading specifically to the final product, and not an enzyme which catalyzes a reaction common to several metabolic pathways it is the enzyme situated at a strategic junction whose activity is inhibited by the final product.

This type of regulation is particularly characterized by the fact that the effector (the substance which activates or inhibits the enzyme) and the substrate of this enzyme are generally not isosteric., i.e. they have no structural analogy (contrary to the situation in competitive inhibition exerted by analogues of the substrate). That is why they are called allosteric effectors, while the term allosteric enzymes denotes the enzymes in which this type of control is observed.

1. General Properties of Allosteric Enzymes:

In connection with aspartate transcarbamylase, some important charac­teristics of the regulation at the level of allosteric enzymes, but it is of interest here to review the main properties.

A. Kinetics of Reactions Catalyzed by Allosteric Enzymes:

In general, allosteric enzymes have special kinetic properties, and when studying the variation of velocity as a function of substrate concentration one obtains, not a branch of an equilateral hyperbola as in the case of most enzymes, but a sigmoid curve. This S-shaped curve reflects a cooperative effect i.e. the fact that at least 2 substrate molecules interact with the enzyme and that the binding of the first molecule facilitates that of the second.

Very often such a cooperative effect is also manifest in the binding of allosteric effectors (see fig. 8-13), which suggests that the binding of the first allosteric activator (or inhibitor) molecule favours the binding of the second. These results already suggest that there are more than one catalytic site and more than one allosteric site per molecule of enzyme and imply the polymeric or oligomeric nature of allosteric enzymes.

We have not indicated where the allosteric effector binds, but our knowledge of the specificity of enzyme-substrate interaction and our observations on the absence of any structural similarity between substrate and effector suggest that the allosteric effectors do not bind to the active sites, but to different sites called allosteric sites.

Considering the sigmoid nature of the curves expressing enzymatic activity as a function of either the substrate, or the allosteric inhibitor (see fig. 8-13), one therefore has a threshold effect when inhibitor concentra­tion increases or when substrate concentration increases.

Below the threshold, an increase of [S] (see fig. 2-12) or [I] does not cause a significant change of velocity but beyond the threshold, velocity varies considerably for a relatively small increase of [S] or [I]. This enables the cell to adjust the enzymatic activity according to relatively small variations of [S] or [I], but occurring in a zone of critical concentration which corresponds to the intracel­lular concentrations of the metabolites involved.

B. Action of Allosteric Effectors:

There are various types of allosteric inhibitors and using the Lineweaver- Burk plot, it is observed that some allosteric inhibitors are of the competitive type and others of the non-competitive type. But contrary to what we have seen while studying the competitive inhibition of conventional enzymes, there is no competition — in the case of allosteric enzymes — between S and I for the active site of the enzyme (because they have no structural analogy).

The two types of inhibitors bind to allosteric sites, dif­ferent from the active sites, as shown by experiments of desensitization and fractionation of sub-units. In the case of a non-competitive al­losteric inhibitor, the binding of I to the allosteric site of an enzyme to give E — I can thus cause a change of conformation which still permits the binding of S to give E —S —I, but 1/Vmax is increased therefore Vmax is lower.

During the binding of a competitive inhibitor to the allosteric site, there is a change of conformation, an allosteric transition, which causes repercussions on the active site to which S can no longer bind. There is a decrease of -1/Km, i.e. an increase of Km, in other words a decrease of the affinity of the enzyme for S. There exists a type of mixed inhibition, charac­terized by an increase of 1/Vmax i.e. a decrease of Vmax, as well as an increase of Km, i.e. a decrease of the affinity of the enzyme for S.

We have seen how the allosteric transition caused by the binding of an activator favours the binding of the substrate. This change of conformation of the enzyme brings about a decrease of Km, i.e., an increase of affinity for S.

The curve representing the kinetics of the reaction can change from the sigmoid form to the hyperbolic form (see curve 2 of figure 2-12), but one must be very cautious because it has been shown in some cases that this change of order of the reaction was only apparent (it was due to an inaccuracy in the first part of the curve which did not show the sigmoid character).

C. Desensitization and Dissociation of Allosteric Enzymes:

Allosteric enzymes can be made insensitive to allosteric effectors, either after a mutation, or in vitro by a physical or chemical treatment: variation of pH, temperature, ionic strength action of urea, mercurial agents, proteolytic enzymes, etc.

This desensitization generally does not affect catalytic activity, which sup­ports the hypothesis of separate catalytic and allosteric sites. It is often reflected by a modification of kinetics which changes from the sigmoid form to the hyperbolic “Michaelian” form. The fact that the enzyme conserves a catalytic activity but is no longer sensitive to the allosteric site was first inter­preted as the sign of an alteration of the allosteric site (by the desensitizing agent) not affecting the catalytic site.

In reality, for the regulatory effects to manifest themselves not only the allosteric sites must be intact and the effectors able to bind to these sites but also, a conformation of the enzyme must be preserved which will enable the allosteric transition and especially the repercus­sion — at the catalytic site — of an event affecting the allosteric site.

In fact, it was observed in some cases, that after desensitization the effector can still bind to the allosteric site on the contrary, due to a modification of the spatial structure of the enzyme, there is disappearance of the cooperative interactions between the various catalytic sites of the same enzyme molecule, between its various allosteric sites and between its catalytic and allosteric sites, thus preventing the allosteric transition.

This allosteric transition consists of a modification of the bonds joining the promoters to one another, which permits the passage of the enzyme from a relaxed state to a constrained state, or vice-versa (see fig. 8-14).

The existence of separate sites for substrate and inhibitor, confirmed ex­perimentally in numerous cases, is particularly evident when it is possible to dissociate an allosteric enzyme in distinct sub-units, some carrying the catalytic sites and others carrying the allosteric sites.

This is the case for example with aspartate transcarbamylase, which is inhibited by CTP and activated by ATP, can be desensitized by heat or urea, but can also be dissociated by mercurial agents: the native enzyme (molecular weight = 310 000) consists of 6 polypeptide chains with catalytic activity (molecular weight = 33 000) and having each a site for the binding of the substrate, and 6 regulatory chains (molecular weight = 17 000) enabling the binding of 6 CTP.

On isolating the catalytic sub-units it is observed that their specific activity (quantity of substrate transformed per unit time, referred to the quantity of protein) is greater than that of the native enzyme, which is not surprising because the elimination of the regulatory sub-units — inactive in the catalysis process — is in a way a purification of the enzyme if one considers only the catalytic point of view.

D. Model of Monod, Wyman arid Changeux:

To explain the phenomena observed during the study of allosteric enzymes, these authors proposed a model whose important characteristics are as follows:

1. The allosteric enzymes are oligomers, whose protomers are associated so that the molecule comprises at least one axis of symmetry (the protomer is defined as the structure which has a binding site for each ligand, i.e. for each substance capable of binding — i.e. substrate, activator and inhibitor — and must not be mistaken for the sub-unit which results from the dissociation of the enzyme and can contain — as in the case of aspartate-transcarbamylase — only one site, catalytic or allosteric)

2. Each protomer possesses only one site permitting the formation of one specific complex with each category of ligand

3. The allosteric enzyme may have different but interconvertible conformations. One often speaks of relaxed state and constrained state. These states are in equilibrium and differ either by the distribution and energy of bonds between the protomers (which determine the constraints imposed oil protomers), or by the affinity of the various sites for the corresponding ligands.

Figure 8-14 shows a simple diagram — with only 2 protomers — to illustrate the model. At first, there is equilibrium between the relaxed form and the constrained form if one of the ligands (e.g., the substrate) has a greater affinity for one of these 2 forms, a relatively small concentration of this ligand will permit the binding of a substrate molecule to a protomer of the form con­sidered, which will shift the equilibrium in favour of this form and will facilitate the binding of the substrate.

But an increase of the concentration of an antagonistic ligand (here the inhibitor) is enough for the equilibrium to be shifted in the reverse direction. Allosteric phenomena are reversible and depend on the concentrations of the various ligands. Such a model explains the fact that a sigmoid curve is obtained when velocity is expressed as a function of [S] or [I].

The diagram of fig. 8-14 is valid for an allosteric enzyme of the K type. In this case, in the absence of substrate, the equilibrium is in favour of the form having a low affinity for the substrate. But as observed above, when [S] in­creases, the equilibrium is shifted in favour of the form having a greater affinity for S.

Conversely, the equilibrium is shifted by the inhibitor in favour of the form having a low affinity for S and the allosteric transition consists precisely in this change of equilibrium. Therefore, the inhibitor decreases the affinity of the enzyme for S (Ks increases), and conversely the substrate decreases the affinity of the enzyme for the inhibitor (K, increases), whence the name K type enzyme.

Other models were proposed to explain the kinetic properties of allosteric proteins. According to the model of induced fit proposed by Koshland, Nemethy and Filmer, there is only one configuration for the protein in the absence of ligand it appears that the binding of the ligand induces a conforma­tional modification of the protomer, which transforms the interactions between the sub-units and changes the catalytic properties.

It appears that the conformation of an enzymatic protein, which we called tertiary and quaternary structure, is not exclusively determined by the primary structure. Actually, it is observed that small molecules (substrates, activators, inhibitors), by binding to specific sites, are capable of causing slight modifications of the spatial structure of the protein.

2. Main Modes of Regulation:

1. Feedback inhibition consists in the inhibition of the first enzyme of a reaction series by the metabolite which is the terminal product of this series. The intracellular concentration of this metabolite therefore controls the rate of its own biosynthesis. In the following we are considering feedback inhibition in straight and branched reaction series.

2. Activation of an enzyme by a precursor of the substrate or by the substrate itself.

3. Activation by a degradation product of the terminal metabolite causing a new increase of the concentration of this metabolite (which may be a high energy potential substance for example).

4. Activation of an enzyme of a metabolic series leading to a metabolite A by a metabolite B, which is synthesized by an independent series, when A and B are both necessary for the synthesis of the same macromolecules, which permits a coordinated production of precursors (in the case of nucleotides).

The activity of an allosteric enzyme can be controlled by several of these regulatory modes. Thus, aspartate transcarbamylase, the first enzyme of the pathway leading to the synthesis of pyrimidine nucleotides, is feedback-in­hibited by a terminal product (CTP), activated by the substrate and also activated by ATP, a ribonucleoside triphosphate required – jointly with UTP and CTP – for the biosynthesis of RNAs.

3. Feedback Inhibition in Straight and Branched Reaction Chains:

A. Feedback Inhibition in Straight Reaction chains:

In straight metabolic sequences, it is generally the first enzyme (E1) which is the regulatory enzyme, i.e. the enzyme subjected to a control of allosteric type. By “first enzyme” one must generally understand the enzyme which catalyzes the first reaction specific of the metabolic pathways con­sidered.

For example, in the case of the biosynthesis of pyrimidine ribo­nucleotides, it is aspartate transcarbamylase which is subjected to feedback control and not an enzyme permitting the synthesis of carbamyl-phosphate or aspartate these two compounds can also enter other metabolic pathways, while their combination to give carbamyl aspartate is really the first reaction leading specifically to pyrimidine nucleotides. The first enzyme of the chain is generally the only one to be inhibited by the final product its activity therefore determines the functioning of the whole sequence of reactions.

The inhibition of this enzyme by the final product of the chain of reactions is of obvious interest. When this final product is in excess, the inhibiting effect it exerts on the first enzyme decreases the rate of this first reaction and consequently restricts its own biosynthesis. Since the series of biosynthetic reactions usually require energy, this regulation process enables the cell to save energy.

This economy is however smaller than the one made through the repression process: when a substance X is in excess, repression enables the cell to dispense with not only the biosynthesis reactions of X, but also the transcrip­tion of genes into mRNA and the translation of the polycistronic mRNA into the enzymes required for the biosynthesis of X, while in feedback inhibition the enzymes required are present but do not function.

On the contrary, feedback inhibition appears as a process more rapid than repression. An excess of a substance X can immediately inhibit the first enzyme of the chain of reactions leading to X, while the effects of repression are manifest only after the disappearance — through catabolism — of the molecules of enzymes and mRNAs existing in the cell (and which are not replaced because the expression of the corresponding genes is blocked).

Feed­back inhibition which is based — as mentioned above — on the phenomenon of allosteric transition, i.e. on the shift of a state of equilibrium in favour of one of the two conformations of the enzyme, is an easily reversible process, very sensitive to small variations of the concentrations of ligands beyond a particular threshold, and is therefore characterized by a great flexibility.

B. Feedback Inhibition in Branched Reaction Chains:

Feedback inhibition poses special problems in the case of branched reactions chains where one could a priori fear that the excess of the final product of one of the branchings would cause — if it inhibits the first enzyme of the chain — the arrest of the synthesis of substances produced by the other branchings, substances which are not necessarily in excess.

To study these problems we will take the example of the biosynthesis of amino acids deriving from aspartate, this will enable us to study their regulation with the help of a simplified diagram.

a) Feedback Inhibition Limited to Branchings:

As shown by figure 8-15, the amino acid which is the final product of a branching can inhibit the first step of the sequence of reactions leading only to its biosynthesis. The biosynthesis of other amino acids is therefore not affected. Lysine inhibits, dihydro-dipicolinate synthetase, threonine inhibits homoserine kinase (HK), methionine inhibits the succinylation of homoserine and isoleucine inhibits threonine deaminase (TD).

b) Iso-Enzymatic Control:

Three aspartokinases (AK) have been iden­tified in E.coli each of them is subjected to a regulation by a specific repression mechanism and two are subjected to a feedback inhibition which is also specific.

Moreover, there are also 2 homoserine dehydrogenases (HSDH) whose regulation is identical to that of the first two aspartokinases, as shown by the table below:

It has been shown that the two catalytic activities AK I and HSDH I are carried by one and the same polypeptide chain, the same is true of the activities AK II and HSDH II. It is evident that the existence, for example in the case of aspartokinase, of 3 isoenzymes whose synthesis and activity are controlled by different terminal products, enables the cell — in case of repression of the biosynthesis or inhibition of the activity of one of the aspartokinases due to a high concentration of one amino acid — to continue to synthesize the other amino acids deriving from aspartate thanks to the other two aspartokinases which are not affected. The existence of 3 isoenzymes to catalyze this first reaction permits an independent regulation by the various terminal products (fig. 8-16).

c) Concerted or Multivalent Feedback Inhibition:

In some organisms of the genus Rhodopseudomonas or Bacillus there is only one aspartokinase which is not affected by the excess of only one of the terminal products (Lys, Thr, Ile), but which is feedback inhibited when there is excess of both lysine and threonine. However this concerted feedback inhibition is not total, which permits the synthesis of methionine.

Other possibilities of control exist in some organisms in this branched chain of biosynthesis of amino acids deriving from aspartate. We cannot study all of them but the types of regulation we examined do show that varied mechanisms were selected by the living organisms in the course of evolution to solve the special problems posed by regulation in the metabolic pathways presenting branchings.


Competitive Inhibition

  • Contributed by Henry Jakubowski
  • Professor (Chemistry) at College of St. Benedict/St. John's University

Competitive inhibition occurs when substrate ((S)) and inhibitor ((I)) both bind to the same site on the enzyme. In effect, they compete for the active site and bind in a mutually exclusive fashion. This is illustrated in the chemical equations and molecular cartoon below.

There is another type of inhibition that would give the same kinetic data. If (S) and (I) bound to different sites, and (S) bound to (E) and produced a conformational change in (E) such that (I) could not bind (and vice versa), then the binding of (S) and (I) would be mutually exclusive this is called allosteric competitive inhibition.

Inhibition studies are usually done at several fixed and non-saturating concentrations of (I) and varying (S) concentrations. The key kinetic parameters to understand are Vm and (K_m). Let us assume for ease of equation derivation that I binds reversibly, and with rapid equilibrium to E, with a dissociation constant Kis. The "s" in the subscript "is" indicates that the slope of the 1/v vs 1/S Lineweaver Burk plot changes while the y intercept stays constant. Kis is also named Kic where the subscript "c" stands for competitive inhibition constant.

The key kinetic parameters to understand are (V_m) and (K_m). Let us assume for ease of equation derivation that (I) binds reversibly, and with rapid equilibrium to (E), with a dissociation constant (K_). A look at the top mechanism shows that even in the presence of (I), as (S) increases to infinity, all (E) is converted to (ES). That is, there is no free (E) to which (I) could bind. Now remember that

[V_m= k_E_o.] Under these condition, [ES = E_o] and [v = V_m.] Hence (V_m) is not changed, but the apparent (K_m) ( (K_)) will.

We can use Le Ch â telier's Principle to understand this. If (I) binds to (E) alone, and not ES, it will shift the equilibrium of (E + S ightleftharpoons ES) to the left, which would have the effect of increasing the (K_) (i.e., it would appear that the affinity of (E) and (S) has decreased.). The double reciprocal plot (Lineweaver Burk plot) offers a great way to visualize the inhibition. In the presence of (I), (V_m) does not change, but (K_m) appears to increase. Therefore, (1/K_m), the x-intercept on the plot will get smaller, and closer to 0. Therefore the plots will consists of a series of lines, with the same y intercept ((1_/V_m)), and the x intecepts ((-1/K_m)) closer and closer to the 0 as (I) increases. These intersecting plots are the hallmark of competitive inhibition.

Note that in the first three inhibition models discussed in this section, the Lineweaver-Burk plots are linear in the presence and absence of inhibitor. This suggests that plots of (v) vs. (S) in each case would be hyperbolic and conform to the usual form of the Michaelis Menton equation, each with potentially different apparent (V_m) and (K_m) values.

An equation, shown in the diagram above, can be derived which shows the effect of the competitive inhibitor on the velocity of the reaction. The only change is that the (K_m) term is multiplied by the factor (1+I/K_). Hence (K_ = K_m(1+I/K_)). This shows that the apparent (K_m) does increase as we predicted. (K_) is the inhibitor dissociation constant in which the inhibitor affects the slope of the double reciprocal plot.

Wolfram Mathematica CDF Player - Competitive Inhibition v vs S (free plugin required)

4/6/14Wolfram Mathematica CDF Player - Competitive Inhibition - Lineweaver Burk(free plugin required)

If the data was plotted as (v_o) vs. (log S), the plots would be sigmoidal, as we saw for plots of (ML) vs. (log,L) in Chapter 5B. In the case of competitive inhibitor, the plot of ( v_o) vs/ (log , S) in the presence of different fixed concentrations of inhibitor would consist of a series of sigmoidal curves, each with the same (V_m), but with different apparent (K_m) values (where (K_ = K_m(1+I/K_)), progressively shifted to the right. Enyzme kinetic data is rarely plotted this way, but simple binding data for the (M + L ightleftharpoons ML) equilibrium, in the presence of different inhibitor concentrations is.

Reconsider our discussion of the simple binding equilibrium, (M + L ightleftharpoons ML). When we wished to know how much is bound, or the fractional saturation, as a function of the log L, we considered three examples.

  1. (L = 0.01 K_d) (i.e. (L ll K_d)), which implies that (K_d = 100L). Then [Y = dfrac<[K_d+L]>= dfrac<[100L + L]>&asymp1/100.] This implies that irrespective of the actual [L], if (L = 0.01 K_d), then Y &asymp0.01.
  2. (L = 100 K_d) (i.e. (L gg K_d)), which implies that (K_d = L/100). Then [Y = dfrac<[K_d+L]>= dfrac<[(L/100) + L]>= dfrac<100L><101L>&asymp 1.] This implies that irrespective of the actual ([L]), if (L = 100 K_d), then (Y &asymp1).
  3. (L = K_d), then (Y = 0.5.)

These scenarios show that if L varies over 4 orders of magnitude ((0.01 K_d < K_d < 100K_d)), or, in log terms, from

[-2 + log , K_d < log, K_d< 2 + log , K_d) ,]

irrespective of the magnitude of the (K_d), that Y varies from approximately 0 - 1.

In other words, Y varies from 0-1 when L varies from log (K_d) by +2. Hence, plots of (Y) vs. (log L) for a series of binding reactions of increasingly higher (K_d) (lower affinity) would reveal a series of identical sigmoidal curves shifted progressively to the right, as shown below.

The same would be true of (v_o) vs. (S) in the presence of different concentration of a competitive inhibitor, for initial flux, (J_o) vs. ligand outside, in the presence of a competitive inhibitor, or (ML) vs. (L) (or (Y) vs. (L)) in the presence of a competitive inhibitor.

Wolfram Mathematica CDF Player - Competitive Inhibition v vs logS (free plugin required)

In many ways plots of v0 vs lnS are easier to visually interpret than plots of v0 vs S . As noted for simple binding plots, textbook illustrations of hyperbolas are often misdrawn, showing curves that level off too quickly as a function of [S] as compared to plots of v0 vs lnS, in which it is easy to see if saturation has been achieved. In addition, as the curves above show, multiple complete plots of v0 vs lnS at varying fixed inhibitor concentration or for variant enzyme forms (different isoforms, site-specific mutants) over a broad range of lnS can be made which facilitates comparisons of the experimental kinetics under these different conditions. This is especially true if Km values differ widely.

Now that you are more familiar with binding, flux, and enzyme kinetics curves, in the presence and absence of inhibitors, you should be able to apply the above analysis to inhibition curves where the binding, initial flux, or the initial velocity is plotted at varying competitive inhibitor concentration at different fixed concentration nonsaturating concentrations of ligand or substrate. Consider the activity of an enzyme. Lets say that at some reasonable concentration of substrate (not infinite), the enzyme is approximately 100% active. If a competitive inhibitor is added, the activity of the enzyme would drop until at saturating (infinite) (I), no activity would remain. Graphs showing this are shown below.

Figure: Inhibition of Enzyme Activity - % Activity vs log [Inhibitor]

A special case of competitive inhibition: the specificity constant: In the previous chapter, the specificity constant was defined as kcat /KM which we also described as the second order rate constant associated with the bimolecular reaction of (E) and (S) when (S ll K_M). It also describes how good an enzyme is in differentiating between different substrates. If has enzyme encounters two substrates, one can be considered to be a competitive inhibitor of the other. The following derivation shows that the ratio of initial velocities for two competing substrates at the same concentration is equal to the ratio of their (k_/K_M) values.


Advances in Radiation Biology

B Specific Binding of RNA Polymerase to Various DNA Templates Is Not Affected Even at Very High UV Doses

Competitive inhibition of synthesis to RNA from nonirradiated DNA by the presence of UV-irradiated DNA was utilized as a measure of the binding of RNA polymerase to UV-irradiated T4 DNA ( Sauerbier et al., 1970 ). These studies employed the following concept: The rate of RNA synthesis with a mixture of DNA templates, one-half of which is nonirradiated and the other half irradiated, should be one-half the sum of the individual rates of RNA synthesis with either template, (RO + RUV)/2, provided that UV irradiation has not altered the binding of RNA polymerase to DNA and that the reaction is saturated with each template. Reduced polymerase binding to UV-irradiated DNA would be indicated by resultant rates higher than (RO + RUV)/2. (Here, RO stands for the rate of RNA synthesis with the nonirradiated DNA template and RUV for the rate observed with the irradiated template.) As Fig. 2 shows, binding of E. coli RNA polymerase to DNA of bacteriophage T4 is not measurably affected by UV doses up to approximately 1000 ergs/mm 2 . This dose is equivalent to about 150 lethal hits to T4vx ( Harm, 1963 ), or about 220 thymine dimers in the early region of the T4 genome ( Sauerbier, 1964 Sauerbier et al., 1970 ), or about three to four phage-lethal hits per T4 scripton comprised of an average of three to four genes ( Stahl et al., 1970 Sauerbier et al., 1970 Hercules and Sauerbier, 1973 O⟺rrell and Gold, 1973 ). Clearly, loss of binding of RNA polymerase to UV-irradiated DNA contributes little, if at all, to the loss of viability of T4.

Fig. 2 . In vitro rates of RNA synthesis with nonirradiated T4 DNA, UV-irradiated T4 DNA and mixtures of nonirradiated and UV-irradiated T4 DNA. Curve A: (•) kinetics of [ 3 H] ATP incorporation with 34 μg/ml unirradiated T4 DNA (○) with 34 μg/ml DNA present at the onset of incubation and additional 34 μg/ml added 8 min later. Curve B: (▪) same as curve A (•) but with 940 ergs/mm 2 irradiated T4 DNA (□) same as curve A (○) but with 940 ergs/mm 2 irradiated T4 DNA. Curve E: one-half the sum of the rates of synthesis with unirradiated (A) and with 940 ergs/mm 2 irradiated T4 DNA (B). Curve C: (Δ) RNA synthesis with 34 μg/ml unirradiated DNA present at the onset of incubation and 34 μg/ml of 940 ergs/mm 2 unirradiated DNA added 8 min later. Curve D: (▴) same as curve C, except that 34 μg/ml of 940 ergs/mm 2 irradiated DNA were added first and the unirradiated 34 Mg/ml were added 8 min later. The specific activity of [ 3 H] ATP was 1 Ci/mole, and the concentration of RNA polymerase 10.15 μg/ml. Ordinate gives the nmoles ATP incorporated in 0.2-ml aliquots. Abscissa gives the time of incubation at 37°C.

From Sauerbier et al. (1970) with permission of Elsevier Publishing Company. Copyright © 1970

Inspection of Fig. 2 shows a resultant rate of RNA synthesis that is actually less than one-half the sum of the rates with either template. This has been interpreted as a slowdown in release of RNA polymerase from the UV-irradiated template DNA and not as an increased binding to UV-irradiated DNA ( Sauerbier et al., 1970 ). Since this interpretation agrees with other observations on the transcription of UV-irradiated DNA [no loss of RNA polymerase binding ( Ishihama and Kameyama, 1967 Chamberlin and Ring, 1970 ), no effect on the rate of RNA chain initiation during the first 10 min of polymerization ( Sauerbier et al., 1970 ), and effective recycling of RNA polymerase ( Michalke and Bremer, 1969 Sauerbier et al., 1970 )], it seems to be correct.

No loss of binding of E. coli RNA polymerase to E. coli DNA has been reported by Ishihama and Kameyama (1967) and no loss of T7 RNA polymerase binding to T7 DNA up to 80,000 ergs/mm 2 was reported by Chamberlin and Ring (1973) . These latter authors argued that the number of polymerase binding sites does not increase as a consequence of UV irradiation to T7 DNA. In contrast to the observations made with UV-irradiated bacterial and bacteriophage DNA, formation of new, unproductive binding sites for E. coli RNA polymerase by UV irradiation of calf thymus DNA has been repeatedly reported ( Hagen et al., 1964 Zimmermann et al., 1965 ). Since the initiation of transcription on calf thymus DNA occurs nonspecifically ( Burgess et al., 1969 ) at single-strand breaks ( Hagen et al., 1970 ), the UV effects on binding of polymerase and on RNA chain initiation with this particular template should not be generalized.

Systematic investigations of RNA polymerase binding to UV-irradiated DNA templates, involving several types of RNA polymerase and several types of DNA templates and covering a wide dose range, are still lacking, although the DNA binding assay to nitrocellulose filters via RNA polymerase ( Jones and Berg, 1966 Hagen et al., 1970 ) should make direct binding observations experimentally quite feasible (at least for RNA polymerases which bind strongly to the DNA template).


Advances in Radiation Biology

B Specific Binding of RNA Polymerase to Various DNA Templates Is Not Affected Even at Very High UV Doses

Competitive inhibition of synthesis to RNA from nonirradiated DNA by the presence of UV-irradiated DNA was utilized as a measure of the binding of RNA polymerase to UV-irradiated T4 DNA ( Sauerbier et al., 1970 ). These studies employed the following concept: The rate of RNA synthesis with a mixture of DNA templates, one-half of which is nonirradiated and the other half irradiated, should be one-half the sum of the individual rates of RNA synthesis with either template, (RO + RUV)/2, provided that UV irradiation has not altered the binding of RNA polymerase to DNA and that the reaction is saturated with each template. Reduced polymerase binding to UV-irradiated DNA would be indicated by resultant rates higher than (RO + RUV)/2. (Here, RO stands for the rate of RNA synthesis with the nonirradiated DNA template and RUV for the rate observed with the irradiated template.) As Fig. 2 shows, binding of E. coli RNA polymerase to DNA of bacteriophage T4 is not measurably affected by UV doses up to approximately 1000 ergs/mm 2 . This dose is equivalent to about 150 lethal hits to T4vx ( Harm, 1963 ), or about 220 thymine dimers in the early region of the T4 genome ( Sauerbier, 1964 Sauerbier et al., 1970 ), or about three to four phage-lethal hits per T4 scripton comprised of an average of three to four genes ( Stahl et al., 1970 Sauerbier et al., 1970 Hercules and Sauerbier, 1973 O⟺rrell and Gold, 1973 ). Clearly, loss of binding of RNA polymerase to UV-irradiated DNA contributes little, if at all, to the loss of viability of T4.

Fig. 2 . In vitro rates of RNA synthesis with nonirradiated T4 DNA, UV-irradiated T4 DNA and mixtures of nonirradiated and UV-irradiated T4 DNA. Curve A: (•) kinetics of [ 3 H] ATP incorporation with 34 μg/ml unirradiated T4 DNA (○) with 34 μg/ml DNA present at the onset of incubation and additional 34 μg/ml added 8 min later. Curve B: (▪) same as curve A (•) but with 940 ergs/mm 2 irradiated T4 DNA (□) same as curve A (○) but with 940 ergs/mm 2 irradiated T4 DNA. Curve E: one-half the sum of the rates of synthesis with unirradiated (A) and with 940 ergs/mm 2 irradiated T4 DNA (B). Curve C: (Δ) RNA synthesis with 34 μg/ml unirradiated DNA present at the onset of incubation and 34 μg/ml of 940 ergs/mm 2 unirradiated DNA added 8 min later. Curve D: (▴) same as curve C, except that 34 μg/ml of 940 ergs/mm 2 irradiated DNA were added first and the unirradiated 34 Mg/ml were added 8 min later. The specific activity of [ 3 H] ATP was 1 Ci/mole, and the concentration of RNA polymerase 10.15 μg/ml. Ordinate gives the nmoles ATP incorporated in 0.2-ml aliquots. Abscissa gives the time of incubation at 37°C.

From Sauerbier et al. (1970) with permission of Elsevier Publishing Company. Copyright © 1970

Inspection of Fig. 2 shows a resultant rate of RNA synthesis that is actually less than one-half the sum of the rates with either template. This has been interpreted as a slowdown in release of RNA polymerase from the UV-irradiated template DNA and not as an increased binding to UV-irradiated DNA ( Sauerbier et al., 1970 ). Since this interpretation agrees with other observations on the transcription of UV-irradiated DNA [no loss of RNA polymerase binding ( Ishihama and Kameyama, 1967 Chamberlin and Ring, 1970 ), no effect on the rate of RNA chain initiation during the first 10 min of polymerization ( Sauerbier et al., 1970 ), and effective recycling of RNA polymerase ( Michalke and Bremer, 1969 Sauerbier et al., 1970 )], it seems to be correct.

No loss of binding of E. coli RNA polymerase to E. coli DNA has been reported by Ishihama and Kameyama (1967) and no loss of T7 RNA polymerase binding to T7 DNA up to 80,000 ergs/mm 2 was reported by Chamberlin and Ring (1973) . These latter authors argued that the number of polymerase binding sites does not increase as a consequence of UV irradiation to T7 DNA. In contrast to the observations made with UV-irradiated bacterial and bacteriophage DNA, formation of new, unproductive binding sites for E. coli RNA polymerase by UV irradiation of calf thymus DNA has been repeatedly reported ( Hagen et al., 1964 Zimmermann et al., 1965 ). Since the initiation of transcription on calf thymus DNA occurs nonspecifically ( Burgess et al., 1969 ) at single-strand breaks ( Hagen et al., 1970 ), the UV effects on binding of polymerase and on RNA chain initiation with this particular template should not be generalized.

Systematic investigations of RNA polymerase binding to UV-irradiated DNA templates, involving several types of RNA polymerase and several types of DNA templates and covering a wide dose range, are still lacking, although the DNA binding assay to nitrocellulose filters via RNA polymerase ( Jones and Berg, 1966 Hagen et al., 1970 ) should make direct binding observations experimentally quite feasible (at least for RNA polymerases which bind strongly to the DNA template).


Inhibitors (Competitive and Non-Competitive)

Enzyme is the digestive system to break down the big molecules to small so it can be used by the cell. Enzyme inhibitors are so important especially in medicine to prevent the molecules to be processed and create bad clinical manifestation for example like allergy

Answer:

competitive inhibitors compete with the actual ligand for the binding site in protein whereas non-competitive inhibitors do not.

Explanation:

inhibitors
is a substance that reduces or decreases the activity of an enzyme. It inhibits the proper functioning of enzyme.

Competitive inhibitors
competitive inhibitors are those which mimics the shape of the actual substrate and binds to the active site.

Figure below explains the functioning, substrate comes and binds to enzyme undergoes product formation and releases the site, making it available for new substrate to come and bind.

when a competitive inhibitor is present which mimics the substrate and binds with the enzyme but is not converted to any product and competes for the enzyme active site with actual substrate.


in simple terms enzymes activity decrease in presence of Competitive inhibitor

in the figure below the enzyme kinetics is low at low concentration of substrate but as the substrate amount increases its activity also reaches back to its normal

Non-competitive inhibitors
Non-competitive inhibitors do not compete for the active site with substrate but does not allow substrate to bind at the active site.

These are actually of two types
1. Allosteric as shown in first figure BELOW, they bind at different position but actually causes change in the active site so new substrate moity cannot bind.
2. in the second figure BELOW the substrate is sterically hindered, blocking the active site so as substrate can not interact with the enzyme.


Figure 1

Figure 2 (sorry couldn't find any better resolution)

in simple terms Non-Competitive Inhibitors do not allow the substrate to bind at the active site.

in the figure below the enzyme activity is low at low concentration of substrate but as the substrate amount increases its activity cannot reach the normal level unlike the competitive inhibitor.


Pharmacodynamics

Elaine M Aldred BSc (Hons), DC, Lic Ac, Dip Herb Med, Dip CHM , . Kenneth Vall , in Pharmacology , 2009

Enzyme Inhibition

Most enzyme receptor sites are not completely specific (there is some structural leeway given the number of combinations possible and the mobility of the protein) and a relatively similarly shaped molecule might be able to achieve a ‘close fit’. This creates competition for molecules of a similar shape and the original molecule might find itself unable to find a binding site because it is already occupied. Many drugs are designed to take advantage of this phenomenon.

The various ways in which enzyme function can be affected are not dissimilar to the ways receptor function can be affected. These principles are worth bearing in mind when looking at chemicals that act directly on receptor sites.

• Competitive Inhibition

Competitive inhibition [ Figure 19.2(i) ] is reversible: another molecule competes with the normal substrate and takes its place in the site.

However, when the normal substrate concentration exceeds that of the competing molecule, the situation is more favourable and the normal substrate replaces the competing molecule.

While the competing molecule is in place it blocks the normal action of the enzyme.

Competitive inhibition can be reversed by increasing the substrate concentration.

• Non-competitive Inhibition

Non- competitive inhibition [ Figure 19.2(ii) ] is reversible.

The inhibitor, which is not a substrate, attaches itself to another part of the enzyme, thereby changing the overall shape of the site for the normal substrate so that it does not fit as well as before, which slows or prevents the reaction taking place.

This type of inhibition decreases the turnover rate of an enzyme rather than interfering with the amount of substrate binding to the enzyme. The reaction is slowed rather than stopped. Non-competitive inhibition, therefore, cannot be increased by increasing the substrate.

• Irreversible Inhibition

The inhibitor becomes covalently linked or bound to the enzyme so tightly that is very difficult to detach it from the enzyme [ Figure 19.2(iii) see Chapter 3 ‘Bonds found in biological chemistry’, p. 13 ].


Watch the video: animus Tutorials: Enzyme - Katalysatoren des Stoffwechsels (August 2022).