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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.
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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:
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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.
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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:
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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.
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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.
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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.
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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.
