Control of Metabolism in Microorganisms

In this article we will discuss about the regulation and control of metabolism in microorganisms.

The biological organisms, specially microorganisms take up nutrients and metabolize them through numerous enzyme-catalysed reactions to build-up their body components as well as extract energy.

All these anabolic and catabolic reactions run in a highly coordinated and harmonious manner to ensure optimal growth and multiplication of an organism. To make this possible, a living organism possesses adequate means to regulate the multifarious biochemical reactions, so that break-down or synthesis of metabolites can be accelerated or slowed down according to necessity. This coordination of various, often complicated, biochemical reactions constitutes regulation of metabolism.

The main objective of metabolic regulation is to enforce a stringent and, at the same time, a meaningful biochemical economy, so that the available nutrients may be utilized for the maximum benefit of the organism.

To achieve this objective, production of any metabolite or an enzyme in quantities more than what is needed may have to be stopped. Similarly, environmental conditions may call for synthesis of some enzyme or enzymes which are either absent or present in a very low concentration. Living organisms possess inborn capacities to make such changes possible through different control mechanisms. These mechanisms are best understood in bacteria.

The regulatory or control mechanisms operate in two levels. In one, the activity of enzymes already synthesized and present in the cell is modulated, i.e. the enzyme activity is either accelerated or diminished (inhibited) according to the need of the cell. These mechanisms operate, therefore, at biochemical level. The other type of control operates at the genetic level. In this case, the synthesis of enzymes may be stopped or geared up.

There are several mechanisms by which the enzyme activity can be modulated. In most cases, a small molecule — which may be a metabolite, or a nutrient molecule present in the environment — acts as the modulator or effector. An effector can be positive or negative, depending on whether it accelerates the rate of reaction of the regulatory enzyme, or inhibits the enzyme concerned.

The evidence of the existence of the control mechanism in organisms came from several observations.

Two of these are briefly mentioned below:

When E. coli bacteria are grown in a medium containing inorganic salts and radioactive glucose (¹⁴C-glucose), all components of the bacteria become labelled with the radioactive isotope. Now, if a non-radioactive amino acid is added in such a medium, it is taken up by the bacteria and incorporated into proteins. As a result, the radioactivity of the particular amino acid in the protein is markedly diminished or becomes nil.

This phenomenon was initially designated as ‘isotopic competition’. This observation suggested that the bacteria possess the ability to stop or slow down a biosynthetic pathway of an amino acid when it is readily available in the medium. The bacteria adopt such a measure to minimize unnecessary expenditure of energy and nutrients.

Another evidence is provided by the excretion of intermediate products by auxotrophic mutants of bacteria. An auxotroph is a defective mutant which is unable to synthesise an essential metabolite. These mutants obligately require the addition of the particular metabolite in the growth medium.

It is observed that when the added metabolite is fully consumed by the bacteria, the precursor on which the mutant enzyme acts is excreted into the medium. This is shown below in a hypothetical pathway in which the precursor A is converted to an essential metabolite through three steps. If the mutant has a single block in the last step, the intermediate product C is excreted.

The inference from this observation is that so long as the metabolite is present in the medium, the bacteria do not produce it from the precursor A and, therefore, excretion of C does not occur. But, as soon as the metabolite is fully exhausted, the bacteria starts the biosynthetic pathway which cannot proceed beyond C, because the enzyme converting C to the metabolite is defective due to mutation. As a result, C accumulates in the cells and is eventually excreted.

The observation suggests that so long a particular essential metabolite is available in the medium, the bacteria can stop synthesis of the metabolite. In other words, a metabolite can regulate its own synthesis according to necessity. A metabolite is generally an end-product of a biosynthetic pathway and can act as an effector or modulator.

Feedback Control:

In most biosynthetic pathways of essential metabolites, a number of enzymes catalyse the different steps sequentially, so that the product of the first enzyme reaction acts as the substrate for the second enzyme and so on, until the last enzyme produces the final product (the end-product).

The end-product of such a sequence generally acts as an effector or modulator of the first enzyme of the pathway. In other words, the end-product feeds back to control the, entire biosynthetic pathway. This is known as feedback control.

As the end-product exerts such control, it is also known as end-product inhibition. This type of control becomes necessary to stop over-production of a metabolite, or to stop production when the metabolite is freely available in the medium.

Feedback control operates in many biosynthetic pathways of bacteria. In all cases, the enzymes which are regulated by feedback mechanism have an allosteric nature. Allosteric enzyme proteins have different sites for binding the substrate and the effector.

The properties of allosteric enzymes will help in understanding the mechanism of feedback control which are as follows:

(a) Properties of Allosteric Enzyme Proteins:

Allosteric enzymes always consist of more than one subunit, generally several. These subunits are protomers, each of which is made from a single polypeptide chain. Allosteric protein are generally larger in size than non-allosteric proteins. Each composite enzyme protein molecule has more than one substrate-binding site and has one or more sites for binding the effector.

The binding of an effector may either enhance the affinity of the enzyme molecule to the substrate or may reduce or abolish the affinity to the substrate. In the first case, the effector acts as an activator and, in the second case, as an inhibitor. Thus, the activity of an allosteric enzyme is profoundly influenced by the effector.

There is an apparent similarity between the inhibition of enzyme activity by a competitive inhibitor (see Enzymes) and that caused by a negative effector, e.g. an end-product producing feedback inhibition. But the two are basically different.

A competitive inhibitor binds to the same site of the enzyme protein as docs the normal substrate, whereas an end-product acting as a negative effector binds to a different site which is distinct from the substrate-binding site.

Moreover, a competitive inhibitor resembles the normal substrate in chemical structure, as a result of which the enzyme molecule fails to distinguish it from the substrate and allows it to enter into the substrate-site. In case of end-product inhibition, the effector has no structural similarity with the substrate.

Another important feature of the allosteric enzymes which distinguishes them from non-allosteric ones is their behaviour towards substrate concentration. Allosteric proteins consist of several protomers. When one of the protomer accepts a substrate molecule, it activates the other protomer or protomers to bind more substrate molecules in their respective substrate-binding sites.

Thus, the catalytic activity of the whole molecule increases due to cooperative effect of the individual protomers. This behaviour of allosteric enzymes differs from normal enzymes which show a Michaelis- Menten kinetic where the velocity of an enzyme reaction increases linearly until all the substrate- binding sites are saturated with substrate.

In contrast, allosteric enzymes exhibit a sigmoid-curve when the reaction velocity is plotted against .substrate concentration (Fig. 8.92). The sigmoid nature of the curve suggests that the binding of the first substrate molecule causes a conformational change of that protomer and this is transmitted to another protomer making it fit to accept another molecule of the substrate. With the increase in substrate concentration, more and more of substrate-binding sites are opened through cooperative effect of the protomers.

The allosteric enzymes not only have binding sites of substrates, but also separate sites for binding of effectors. According to the model proposed by Monod and Changeux, an allosteric protein may exist in one of the two alternative conformation states. One of these is the active or catalytic state and the other is the inactive or inhibited state.

Binding of a negative effector (inhibitor) leads to stabilization of the inactive conformation having either no or low affinity for the substrate. On the other hand, binding with substrate leads to an active conformation which has no or low affinity for the effector. The two states are reversible and depend on relative concentrations of the substrate and the inhibitor.

This is schematically represented in the diagram (Fig. 8.93):

(b) Feedback Control of Pyrimidine Nucleotide Biosynthesis:

Pyrimidine biosynthesis begins with condensation of earbamyl phosphate and aspartic acid to yield carbamyl aspartate catalysed by aspartate transcarbamylase (ATCase) which is the first enzyme of the pathway. The pathway ultimately leads to the formation of cytidine triphosphate (CTP) which is thus the end-product of the pyrimidine nucleotide biosynthetic pathway. CTP has been found to inhibit the enzyme ATCase by feedback mechanism, both in vivo and in vitro.

The in vivo effect of CTP on ATCase was elegantly demonstrated through an experiment using a uracil-requiring auxotrophic mutant of E. coli. This mutant excreted considerable amount of carbamyl aspartate in the medium when the supplied uracil was used up. The mutant was unable to synthesise uracil due to a mutational block in the enzyme dihydroorotase which catalyses conversion of carbamyl aspartate to dihydroorotic acid.

Some essential steps of the pathway are given below showing the mutational block in the E. coli mutant:

The mutant E. coli excreted carbamyl aspartate, because it was not converted to dihydroorotic acid due to the defective enzyme, dihydroorotase. That uracil itself is not the inhibitor of ATCase, but that it is CTP, was demonstrated by adding an inhibitor, diazo-oxonorleucine (DON). In presence of DON, ATCase was not inhibited even when sufficient uracil is present.

This inhibitor prevented conversion of UTP to CTP in the biosynthetic pathway. As a result, carbamyl aspartate excretion continued in presence of uracil, and ATCase remains active, because CTP synthesis is inhibited by DON. Only CTP inhibited ATCase activity by feedback inhibition of the first enzyme of the sequence.

The inhibitory effect of CTP on ATCase could also be demonstrated in vitro with isolated ATCase. Purified ATCase can be dissociated into two types of subunits which constitute the allosteric ATCase molecule. One subunit possesses the catalytic activity which converts aspartic acid and carbamyl phosphate to carbamyl aspartate. This subunit is not inhibited by CTP.

The other subunit has no catalytic activity, but can bind CTP. Thus, the two subunits have different functions and the whole ATCase molecule has different sites for binding substrate and the effector (CTP). Hence, ATCase is an allosteric enzyme. In a reaction mixture containing the substrates (aspartic acid and carbamyl phosphate), purified ATCase can catalyse formation of carbamyl aspartate, but in presence of CTP the enzyme activity is inhibited, because it binds to one subunit of ATCase and inhibits its function.

(c) Feedback Regulation of Amino Acid Biosynthetic Pathways:

Regulation of amino acid biosynthesis is an essential feature of microorganisms, because these compounds are the building blocks of proteins and are also required for synthesis of other cellular ingredients. Regulation is necessary for conservation of these valuable compounds by preventing unwanted production.

Feedback control plays a major role in this regulation. The other method of regulation of amino acid biosynthesis is through repression of enzyme synthesis which operates at genetic level.

Feedback inhibition of amino acid biosynthesis was first reported in the isoleucine pathway. It may be recalled that isoleucine is synthesized from another amino acid, threonine. Both these amino acids, along with several others, belong to the aspartic acid family. Threonine acts as a precursor for isoleucine synthesis. The first enzyme, threonine deaminase, catalyzing conversion of threonine to α-keto butyric acid, is inhibited by isoleucine.

Biosynthesis of amino acids often occur through complicated branched pathways. This makes regulation by feedback control a complex process. It has been mentioned at the outset that the end- product of a sequence regulates the first enzyme.

In a branched pathway, if the first enzyme is inhibited by any one of the several end-products, biosynthesis of all the end products of the pathway would be stopped. This would prove detrimental to the organism, because in absence of the other end products, the growth will cease.

The problem can be visualized from the schematic representation of a hypothetical branched pathway:

Starting from a common precursor A, three different end-products (Y), (E) and (R) are synthesized in a branched pathway. If the particular organism is supplied with one of the end-products, say (Y) in the medium, the organism would try to stop synthesis of (Y) by feedback.

In case the end-product (Y) inhibits enzyme I — which happens to be the first enzyme of the branched pathway — synthesis of (E) and (R) will also automatically stop, resulting in cessation of protein synthesis and, eventually, growth. This catastrophe can be avoided if the synthesis of the individual end-products is regulated independently of each other. Microorganisms possess several different means to solve this complicated problem.

Firstly, if the first enzyme of the sequence, e.g. the enzyme I of the hypothetical sequence, is present in more than one molecular species, i.e. isoenzymes or isozymes (see Enzymes for details of isozymes), each isozyme may be sensitive to feedback inhibition by a single end-product.

The other isozymes may still remain active and catalyse conversion of A to B, although the rate of conversion will be slowed down. This type of regulation through isozymes operates in the initial reaction of the biosynthetic pathway of amino acids of the aspartic acid family. The first reaction resulting in phosphorylation of aspartic acid is catalysed by aspartokinase which is present in three isozymic forms in E. coli, each being inhibited by a different end-product.

Secondly, the problem can be tackled if each end-product can regulate only its own synthesis, without affecting the production of other products. This can be achieved if the branch-point enzymes are regulated, e.g., in the above hypothetical example, (Y) may control the enzyme 8, (E) enzyme 4 and (R) enzyme 5, respectively. This type of regulation does occur in branched pathways.

Thirdly, the feedback inhibition may show at least two types of variations. In one of these, known as multivalent feed-back inhibition, the inhibition of the first enzyme of a branched pathway becomes effective only when all the end-products are present in excess to what is needed by the organism. In other words, no individual end-product can enforce inhibition.

A second variation, called cumulative feedback inhibition or co-operative feedback inhibition, involves only partial inhibition of the initial enzyme by any one end-product. Complete inhibition requires the cumulative or cooperative inhibitions of all the end-products. Thus, if there are three end-products, each may exert one-third inhibition of the enzyme concerned.

The different types of feedback inhibitions are schematically represented in Fig. 8.94:

Feedback regulation of branched pathways for amino acid biosynthesis can be best illustrated by that of aspartic acid family and aromatic amino acids.

These are diagrammatically shown in Fig. 8.95 and Fig. 8.96, respectively:

Regulation of Enzyme Activity by Adenosine Phosphates:

Besides feedback mechanism, there are also other means of regulation of enzyme activity. One of these is by regulation of the energy-status of the cell. Living cells possess subtle regulatory mechanisms by which they can control the rate of energy production and energy consumption.

The energy-status of a cell in a given moment has been designated as its energy-charge. When the total adenosine phosphates, i.e. AMP + ADP + ATP, are present in the form of ATP alone, the energy-charge of the cell has a value of 1.0. On the other hand, when all the adenosine phosphates are in the form of AMP, the energy-charge is taken as 0. In the metabolic steady-state, none of these extreme values is attained, because the ATP generation and ATP consumption run concurrently.

When the energy-charge falls below a certain limit, ATP generation is automatically stimulated until the energy-charge reaches a threshold value. This balance of ATP generation and ATP consumption is secured through several regulatory enzymes. These enzymes occur in catabolic pathways.

Regulation of two such pathways, viz. glycolysis (EMP) and TCA cycle, are discussed below:

(a) Regulation of the Glycolytic Pathway:

Evidence that ATP can regulate the glycolytic pathway is supported by the observation that the uncoupling-agent — 2, 4-dinitrophenol (2,4-DNP) which dissociates or uncouples ATP synthesis from electron-transport via the electron-transport system (ETS) can regulate the rate of glucose break-down. Normally, yeasts consume much larger amount of glucose through glycolysis when they grow anaerobically than when they grow aerobically.

This observation first made by Pasteur is known as the Pasteur-effect. A clue to the underlying mechanism of the Pasteur-effect was obtained from the observation that when electron-transport and ATP generation by oxidative phosphorylation are uncoupled by 2, 4-DNP, Pasteur-effect was abolished.

In other words, because yeasts produce much more ATP from the same amount of glucose when they grow aerobically than when they grow anaerobically, the rate of glycolysis is slower in presence of oxygen. In presence of 2, 4-DNP, no ATP is generated by oxidative phosphorylation and, therefore, the amount of ATP is the same as that of anaerobic growth produced by substrate-level phosphorylation alone. The rate-regulating step of glycolysis has been recognized as the conversion of fructose 6-phosphate to fructose 1, 6-bisphosphate catalysed by the enzyme phosphofructokinase.

Phosphofructokinase is an allosteric enzyme which can bind not only its substrate, fructose 6-phosphate, but also ATP which acts as a negative effector. It is also inhibited by citrate and activated by ADP and inorganic phosphate. Thus, it is a multivalent regulatory enzyme.

When the cell has an excess of ATP, the enzyme activity is inhibited, thereby slowing down the rate of glucose break-down. On the other hand, when the cell consumes ATP rapidly, resulting in accumulation of ADP and Pi, the enzyme is activated to restore the energy-charge of the cell to a favourable status.

Thus, the activity of phosphofructokinase can be modulated by the relative concentrations of ATP and ADP. The enzyme is also inhibited by citrate which is the product of condensation of oxalacetic acid and acetyl-CoA originating from decarboxylation of pyruvic acid, the end-product of glycolysis.

Accumulation of citrate in the TCA cycle acts as. a signal to indicate that the electron-transport system is saturated and, therefore, further ATP generation is not immediately necessary. To regulate unnecessary ATP production, citrate inhibits the regulatory enzyme — phosphofructokinase. So feeding of acetyl-CoA into the TCA cycle is slowed down.

(b) Regulation of the TCA Cycle:

TCA cycle is the main energy-generating pathway in which the acetyl-rest of acetyl-CoA is introduced into citric acid and oxidized to CO₂; the H-atoms are transferred to NAD or FAD. NADH₂ and FADH₂ produced in the TCA cycle are re-oxidised via electron transport chain and ATP is generated.

Like the glycolytic pathway, TCA cycle is regulated by the energy status of the cell. The main rate regulating enzyme of the TCA cycle has been identified as NAD-linked isocitrate dehydrogenase. This enzyme catalyses oxidative decarboxylation of isocitric acid to α-ketoglutaric acid with release of CO₂

Isocitrate dehydrogenase is, as expected, an allosteric enzyme which binds in addition to its substrate, isocitric acid, other ligands like ATP and NADH₂, both of which act as negative effectors. On the other hand, ADP acts as a positive effector for this enzyme.

This means that, in presence of excess of ATP or NADH₂, the enzyme activity of isocitrate dehydrogenase is strongly inhibited. In contrast, when ADP binds to the enzyme, its affinity to bind the substrate, i.e. isocitric acid is increased. The net result of these interactions is that excessive break-down of ATP to meet the demand of cellular activity produces ADP to reach a high concentration.

This means that the energy-charge of the cell falls to a lower value. ADP accumulated in the cell immediately gears up the activity of isocitrate dehydrogenase to bind isocitric acid, thereby accelerating the TCA cycle so that more fuel in the form of NADH₂ / FADH₂ is supplied to ETS for ATP generation. Conversely, when excess of ATP is present, it inhibits the enzyme, thereby slowing down the TCA cycle, so that less fuel is supplied to ETS for ATP generation.

Regulation of Enzyme Activity by Chemical Modification of the Enzyme:

Some of the enzyme molecules themselves may be chemically modified from an active state to an inactive one and vice versa, through the action of other enzymes. Such enzymes in the animal system are the phosphorylase which degrades glycogen and the glycogen synthetase which synthesizes glycogen. In bacterial organisms, e.g. E. coli, the glutamine synthetase is an enzyme which is regulated by chemical modification.

Glutamine synthetase is a structurally complex allosteric protein which catalyses the following reaction:

Glutamic acid + NH₃ + ATP –> Glutamine + ADP + Pi

The mechanism of such regulation in E. coli is briefly discussed below:

In E. coli, the enzyme has an important regulatory role, because glutamine serves as a precursor in the synthesis of a number of important compounds. The enzyme is present in two forms — an active form known as glutamine synthetase a and an inactive from known as glutamine synthetase b.

The inactive form is derived from the active form by adenylation i.e. AMP from ATP is added through the action of an adenylating enzyme. When E. coli cells possess excess of glutamine, the adenylating enzyme is activated and glutamine synthetase a is converted to the inactive glutamine synthetase b. In this reaction, glutamine acts as a positive effector (activator) for the allosteric enzyme.

The reverse reaction, i.e. conversion of inactive glutamine synthetase b to the active form is catalysed by a de-adenylating enzyme which, by addition of water, releases AMP and the active form of the enzyme (Fig. 8.97). In this reverse reaction, α-ketoglutarate acts as a positive effector. α-Ketoglutarate is a TCA-cycle intermediate from which glutamic acid is produced by the action of the enzyme glutamic acid dehydrogenase.

Thus, in E. coli, when excess of glutamine is present in the cells, further glutamine synthesis is inhibited automatically by glutamine itself. Similarly, when α-ketoglutaric acid is present in excess, it promotes conversion of glutamic acid to glutamine by the glutamine synthetase reaction.

In E. coli, NH₄⁺-ions in the medium acts as a controlling factor in the inactivation of glutamine synthetase because NH⁺-ions are used for both glutamic acid synthesis and glutamine synthesis.