Why should metabolic pathways be regulated?

This can be a fundamental question at this point of

time in your mind. Let us try to answer this question. There can be 2 broad purposes for the
metabolism of the main energy yielding materials to be regulated.

1. To respond to energy production needs as energy expenditure varies.

2. To respond to physiological needs – pathway need to work in different directions after a
meal when metabolites are being stored as compared with intervals between meals when
storage metabolites are being utilized. The needs are different again in prolonged starvation
and in diabetes mellitus where carbohydrate metabolism is abnormal.

There is another reason that is less self-evident – the potential problem of futile cycles. If
unchecked, this cycle of events proceed and achieve nothing more than to rapidly destroy ATP with
the generation of heat. Extending this to the whole of glycolysis and gluconeogenesis, their
pathways could constitute a giant futile cycle, doing nothing but uselessly destroying ATP.

The same applies to glycogen synthesis and breakdown, and fat synthesis and breakdown, or for
that matter, to virtually any synthesis and breakdown pathways.

Fig: potential large scale futile cycles if metabolism were not controlled.

Therefore the breakdown and synthesis directions of a metabolic pathway must be controlled in a
reciprocal manner – activation of one and inhibition of the other. Independent control of the 2
direction is possible at irreversible metabolic steps. It is here that there are separate reactions in
the 2 directions catalysed by distinct enzymes that can be separately controlled. A freely reversible
reaction is catalysed by the same enzyme in both directions. Substrate cycles are crucially
important control points since they provide the opportunity for independent controls on the
forward and reverse metabolic pathways. Substrate cycling is not completely wasteful. However, a
small amount of cycling may be advantageous in making control on the forward and backward
metabolic pathways more effective. Suppose you have a pathway in which A is converted to C via B
and the reaction A ----- B occurs via a substrate cycle.
The rate of conversion of A to C can be reduced by inhibiting enzyme 1 or activating enzyme 2. If
you do both, the control is much more effective, ie complete shut down. The same type of
considerations apply to increasing the flux of metabolite from A to C. The F-6-P to F-1,6 BP cycle
has dual controls of the type described.

Under some circumstances (sometimes referred to as the futile cycle), fructose-1,6-biphosphate

may be hydrolyzed to form fructose-6-phosphate and phosphoric acid nearly as rapidly as it is
formed. When this is happening, the major effect noticed would be:

Increased temperature in the tissue and organism

A futile cycle occurs when two metabolic pathways run in opposite directions at the same time. The
reaction will have no effect except wasteful dissipation of energy in the form ATP hydrolysis and
heat.it was called futile cycle because of no apparent net utility of this pathway to an organism.
Interconversion of fructose 6-p to fructose 1,6 Bp is an example of futile cycle.

Currently futile cycle is assumed to have a role in metabolic regulation. A very small change in the
activity of an enzyme can have dramatic effect on the overall reaction. For this reason, it is called
substrate cycle. Apart from having a role in metabolic regulation, the cycle is known to generate
heat, and may be used to maintain thermal homeostasis in brown adipose tissues of young
mammals. It is also found to be occurring in insect flight muscle and hibernating animals as they
move from hibernating period to active period.

The regulation of metabolism is concerned primarily with modulation of key reactions that
determine the fluxes of metabolites throughout the various pathways. Modulating the reaction
rate is achieved mainly by control of the activities and concentration of the enzymes that catalyze
key reactions. The enzymes of metabolic pathways do not all operate at their maximum rates.
Rather, the rates of key regulatory enzymes are controlled by the concentration of either the final
end product of the pathway or by a critical intermediate. The regulatory enzymes control the rate
of the entire metabolic pathway. The growth and maintenance of a cell requires a highly integrated
coordination of anabolic and catabolic processes. The regulation of a metabolic pathway occurs at
several ways. Since the functioning unit of a metabolic pathway is the enzyme catalyzed reactions,
metabolic pathways are regulated through control of enzymes. An enzyme can be controlled at two
different levels; its synthesis can be controlled and its activity can be controlled.
Now let us look at controlling the synthesis (monofunctional pathways). In other words, it is
controlling the amount of enzyme present at any given time in the system.

What is true about metabolic pathways

All Enzymes of metabolic pathways do not operate at their maximum rate

A. Regulation of enzyme concentration

1. Substrate induction of enzyme synthesis

The concentration of any given enzyme is determined by the rate of its synthesis and by the rate of
its destruction. The concentration of an enzyme is primarily determined by the rate of specific gene
expression. In the case of catabolic pathways two classes of enzymes are recognized:

(a) constitutive enzymes whose concentration is more or less independent of the presence of their
substrates

(b) inducible enzymes that are not normally produced unless their immediate substrate is present.
Often the presence of the first substrate in a catabolic pathway results in the simultaneous
synthesis of all enzymes involved in its catabolism. This phenomenon is called simultaneous
induction.

2. Catabolic repression

Sometimes substrate induced synthesis of catabolic enzymes is repressed when the energy and
carbon requirements for growth are amply supplied by a different catabolic process. Eg; when
glucose is supplied in addition to a second substrate whose catabolism is under induced enzyme
control the glucose will be utilized preferentially and as long as it is present the enzyme required
for the catabolism of the second substrate will be formed. This repressive effect of glucose on
induced enzyme synthesis is called glucose effect.

3. Feedback repression of enzyme synthesis

Two kinds of end product control are established, namely feedback inhibition and repression. In
the first, the end product exerts a restraining action through inhibition of the enzyme that catalyses
the first step in the biosynthetic pathway. Negative feedback inhibition: here the committing step
of a metabolic pathway is often controlled by an end product of the pathway. It has the following
advantages:
 (i) it prevents the excess accommodation of both products and metabolic intermediates.
(ii) The committing step normally involves the hydrolysis of ATP. Control of the committing
step prevents wasteful depletion of cellular energy supply.

In the case of repression, accumulation of the end products leads to inhibition of the synthesis of
one or more or all of the enzymes involved in its biosynthetic pathway. As in the case of enzyme
induction, when several or all of the enzymes in a given biosynthetic pathway are closely located in
a single operon, they may be susceptible to coordinate repression. Eg; regulation of histidine
biosynthesis in salmonella typhimurium. In this case all the ten enzymes are continued in a single
operon whose expression is subject to regulation by histidine. As a consequence, in normal cells
the synthesis of all ten enzymes in the histidine pathway varies simultaneously and to an identical
degree in response to fluctuations in the concentration of histidine.

Which enzymes in metabolic pathway are regulated? The answer is those enzymes designed by
evolution to be regulated. There are specific regulatory enzymes and are strategically placed. There
are four main ways in which the catalytic activities of strategic enzymes are modulated.

1.      The synthesis and degradation of the enzyme responsible for the committing step can be
controlled.

2.      By covalent modification of protein mainly by phosphorylation and dephosphorylation.

3.      The steady state concentration of the metabolite involved in the committing step can be
controlled by an allosteric enzyme.

4.      The availability of substrate can be controlled through regulation of membrane transport.

Metabolic activities of cells are controlled also by circulating hormones and neural inputs that
operate in the body as a whole according to current needs.

In the previous section, we had elaborately looked at how the synthesis of an enzyme can be
controlled. The degradation of enzyme can be understood by looking at the half life of a protein.
Balance between enzymes synthesis and degradation

Metabolic control in animals by changes in enzyme levels is important. The control is a relatively
long term affair with effects being seen in hours or days rather than seconds. It is at the level of
adaptation to physiological needs. There are many instances where enzyme levels are adjusted. Eg;
the amount of lipoprotein lipase in capillaries is adjusted to the fat demands of the tissues. It’s level
increases in mammary glands. The liver changes its enzyme content within hours in response to
dietary changes – whether it has to cope up with high fat or high carbohydrate intake. In the well
fed state, hepatic enzymes involved in fat synthesis increases in amount, while a few hours of
starvation reverses this situation. Intake of foreign chemicals such as drugs results in a rapid
increase in hepatic drug metabolizing enzymes. The reversal of increases in enzyme levels occur
relatively slowly since the levels are returned to lower values only by destruction of the proteins
( or dilution by cell growth in bacteria) and this is related to their half life. The table below gives an
idea about the half life of certain enzymes.

Enzyme Range
(hours)
Ornithine decarbohylase 0.2
RNA Pol-I 1.3
Tyrosine 2.0
aminotransferase
Serine dehydratase 4.0
PEPCarboxylase 5.0
Aldolase 118
GADPH 130
Cytochrome b 130
LDH 130
Cytochrome c 150

Concentration of an enzyme is regulated by

The type of amino acids present on the amino terminal of an exzyme

day to day metabolic demand

vary from different developmental stages

Remedy
 Animal protein undergo continual synthesis and degradation. The half life some of them is only a
few hours. The prevailing concentration of a given enzyme is determined by both the kinetics of its
formation and of its degradation. The fluctuations of concentration that occur in response to
nutritional and hormonal factors are related to specific effects on either the rate of synthesis or of
degradation. Eg; the increase of live arginase concentration caused by feeding a high protein diet is
solely the result of an increase in the rate of arginase synthesis.

The level of a protein in a cell can be changed either by altering the rate of its production or the
rate of its destruction. Proteins are relatively short lived. The half life of enzymes in liver might
range from about an hour to several days.

which of the following best describes protein turn over?

The balance between protein breakdown and build up

Other regulatory mechanisms

Enzymes are responsible for metabolism and therefore metabolic control involves control of
enzyme activities. There are usually one or more reactions in which there is a large free-energy
change, and these reactions “pull” the P.W. to completion. Such 1st step is called a committing
step. This committing step can be controlled at 3 levels:

1.      Allosteric control

2.      Covalent modification

3.      Miscellaneous factors

Allosteric control of enzyme

It is of central importance in metabolic regulation. These enzymes have one or more binding sites
other than for substrate. Any small molecules that reversibly binds to a site on a protein is called a
ligand. The ligands that bind to the allosteric sites are called allosteric effectors or moderators.
They need not have any structural relationship to the substrates of the enzyme. At a given
substrate concentration, a positive effector increases the activity of the enzyme when it combines
at the allosteric site and a negative effector decreases it.

In a few cases, allosteric effector reduces the catalytic rate of the enzyme so that the Vmax is
reduced. This is very uncommon as compared with the type in which the allosteric effectors alters
only the affinity of the enzyme for its substrate. As mentioned before, enzymes work at sub
saturating levels of [S], increase in their affinity will increase their activity and decrease in their
affinity has the reverse effect. At saturating levels of [S], the Vmax is unchanged by the allosteric
effector even if the affinity is changed, but this situation does not usually occur in the cell.

Allosteric enzymes have multi subunit structure. The reaction profile is sigmoidal rather than
hyperbolic. When an allosteric activator (positive modulator) binds to the enzyme, the s shaped
curve is moved to the left, and with an allosteric inhibitor, to the right. The former is due to
increases in the binding affinity of the enzyme for its substrate; and the latter to a reduction.
The sigmoidal shape of the response to substrate concentration means that over a range of
substrate concentrations, the rate of enzyme catalysis is more sensitive to substrate concentration
change (and therefore, binding affinity change) than with a MM type of enzyme (the curve is
steepest there).

Allosteric control is instantaneous – both in its application and reversibility. The allosteric ligand
attaches to its site by noncovalent bonds and when the concentration of ligand is reduced, it
dissociates from combination with the enzyme and everything goes into reverse.

The vital point about allosteric control is that the allosteric effector need not have any structural
relationship whatsoever to the substrate of the enzyme regulated or even belong to the same area
of metabolism. i.e., the metabolite of one pathway can be regulator of another. Moreover, an
enzyme can have allosteric site for more than one effector and thus can receive control signals
form several metabolic areas, which increases the flexibility of control. Monod describes allosteric
enzymes as the second secret of life (DNA being the first).

Feedback Regulation of multifunctional pathways

The regulation of linear biosynthetic pathways such as biosynthesis of histidine presents no serious
problems. Inhibition of the first step in the pathway by the ultimate end product provides an
elegant control system. On the other hand, regulation of a branched biosynthetic pathway presents
a special problem since a part of the biosynthetic sequence, the conversion of A to C is involved in
the synthesis of both end products E and G. If the first common step is subject to independent
feedback control by either or both of the ultimate end products, a situation could arise in which an
excess of one end product would inhibit the conversion of A to B and thereby cause a deficiency in
the production of the other. A number of fundamentally different mechanisms of regulation are
utilized to avoid such difficulties. Some of them are:

Here one of the intermediates can be converted into two or more products. If one of the end
products completely inhibits the 1st step, it will restrict the formation of the other end product.
There are several mechanisms to control branched pathways.

Enzyme multiplicity or parallel enzymes

Here two different enzymes, acting in parallel, catalyse the 1st reaction in a branched P.W. The
1st enzyme is inhibited by one end product of the branched path, and the 2 nd enzyme by the other
end product. Each end product serves as negative feedback inhibitor of its own formation, but its
synthesis is not curtailed by formation of the other products. An excess of compound E can
therefore inhibit only that fraction of step 1 activity that is catalysed by the E-sensitive enzyme. The
residual step 1 activity resulting from the G-sensitive enzyme would still be available for the
production of compounds B and C needed for the continued synthesis of compound G. In order to
prevent further utilization of B and C for the synthesis of E, there is a second feedback control of
step 3 by the end product. Finally when both the end products are in excess the entire pathway
becomes inoperative

Branched pathways may also be regulated at the 1st step after a branch point. Here, the 1st step that
is unique to each pathway is inhibited. Biosynthesis of aromatic amino acids, purine biosynthesis

Feedback term refers to

Effect of end product on rate reaction

Sequential feedback inhibition

Here the first common step is catalyzed by a single enzyme that is not susceptible to feedback
control by the last common intermediate C, in the branched pathway. In addition, the first
divergent steps c and d are under feedback control by the ultimate end products of their unique
pathways. Eg; biosynthesis of aromatic amino acids in B subtilis.

Here the branch point metabolite inhibits the 1st committing step by negative feedback inhibition.
The inhibition is sequential because accumulation of E causes a decrease in the rate at which C is
converted to D. C, therefore, accumulates and inhibits its own formation by negative feedback
inhibition.
Concerted feedback inhibition
It is also called multivalent feedback inhibition because neither of the ultimate end products alone
is able to affect the first common step in the pathway, but when both are in excess simultaneously
they act in concert inhibit the activity of that enzyme. When the branch point intermediate of a
branch pathway inhibits the 1stcommitting step and when a combination of end products act
together to inhibit the 1st committing step, it is called concerted feedback inhibition. Only the action
of E and F in concert can inhibit the 1st committing step; either one alone has no effect on the
enzyme that catalyses the conversion of A to B. An example is found in Rhodopseudomonas
capsulate. It has only one aspartokinase that is insensitive to feedback inhibition by any one of the
end products of aspartate metabolism. However, when the two end products, threonine and lysine
are both present in excess they inhibit aspartokinase activity.

Synergistic feedback inhibition.

The end products of the branches inhibit the 1st committing step of the pathway and the total
inhibition caused by the mixture of the products is greater than the sum of the inhibitions caused
by each product acting separately. The inhibition is therefore synergistic. The end products also
inhibit their own formation by inhibiting the 1st reaction after the branch point. It differs from the
concerted mechanism only in that each of the ultimate end products has a slight ability to act
independently of the other. However, when excesses of both are present simultaneously the total
inhibition is much greater than the sum of their independent activities. Such action is found in the
regulation of glutamine synthetase activity in bacillus lichiniformis. This enzyme is only slightly
inhibited by low concentrations of either AMP, histidine, or glutamine; however, mixtures of AMP
plus histidine or of glutamine plus histidine cause almost complete inhibition of glutamine
synthetase activity.
Cumulative feedback inhibition
In some cases, a high concentration of any one of the products of a branched pathway partially
inhibits the 1st committing step of the sequence. In fig, D and F each partially inhibits the conversion
of A and B. each inhibitor acts independently of the others and the effects are cumulative. The
presence of one does not influence the activity of the other. Eg; glutamine synthetase. Glutamine is
a source of nitrogen for the biosynthesis of a variety of end products including AMP, CTP,
glucosamine-6-P, histidine, and carbonyl phosphate. Glutamine synthetase is an enzyme catalyzing
the first common step in the biosynthesis of a large number of different end products. Its activity is
inhibited by each one of the six above-end products and also by glycine and alanine. When two or
more inhibitors are present at the same time, their effects are cumulative, and when all eight are
present their activity is completely inhibited.
Which of the following mechanism is allosteric regulation of enzyme activity?
conformational change of enzyme molecule

Covalent modification
There are many cellular regulatory agents which exerts their effect through a protein
phosphorylation system. Phosphorylation is one of the more than hundred known covalent
modifications of proteins. Many of them act through a 2ndmessenger.

The principle is very simple. Enzymes called protein kinases transfer phophoryl groups from ATP to
specific proteins. When this happens, the target enzyme undergoes a conformational change and
change its activity. To reverse the process there are proteins phosphatases that hydrolyse the
phosphate from the protein.

Post-translational covalent modification is an important factor in the

regulation of the enzymes' activity. Choose the mechanism of regulation of
glycogen phosphorylase and glycogen synthetase activities from the
following:
Phosphorylation-dephosphorylation

Miscellaneous factors
Regulation of enzyme activity by Ca²+ / calmodulin:

Many biological reactions are triggered by transient increase in the calcium ion concentration. E.g.
glycogen and lipid degradation release of chemical transmitters by nerves muscle contraction, cell
division, etc. All eukaryotic cells contain calmodulin which modulate cyclic nucleotide
phosphodiesterase, brain adenylate kinase, myosin light chain kinase, phospholipase A2, plant NAD
kinase, etc.

Role of proteolysis in regulation

The best example is the conversion of pepsinogen to pepsin. The activation process is an
autocatalytic process involving limited proteolysis of the inactive precursors and is catalysed by
their respective active derivatives.

Which mechanism of proenzyme conversion into the active enzyme?

partial proteolysis of enzyme molecules.

Adenylate Energy Charge

Adenylate Energy Charge and Phosphorylation state ratio:

ATP, ADP, AMP are allosteric effectors for many enzymes. Their concentration also reflects the
energy state of the cell. A cell is a fully charged when the adenylate system consists of only ATP and
is completely discharged when it consists only of AMP and Pi.

The adenylate energy charge is defined as

                  =          [ATP] + ½ [ADP]

                              ˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉˉ          

                              [AMP] + [ADP] + [ATP]

When the energy charge is high, ATP inhibits the relative rates of a typical ATP generating
(catabolic) pathway and stimulates the typical ATP utilising (anabolic) pathway. ADP is considered
half charged because it has one half as many phoshoanhydride bonds as ATP. The AEC varies
between 0 when only AMP is present and 1.0 when only ATP is present. AEC plays a key role in
metabolic regulation. In most cells, the energy charge is between 0.7-0.9. This is also the region
which the slopes of the curves are steeper. Hence, a small change in the energy charge has a large
effect on the rates of synthetic and degradative pathways in the region of the energy change
between 0.7-0.9. The control of these pathways is designed to maintain energy charge within
narrow limits (buffer). In the plot of the reaction rates of such pathways versus energy charge the
curves are steep near an energy charge of 0.9, where they usually intersect.

An alternative index of the energy status is the phosphorylation potential.

Phosphorylation potential =    [ATP]

                                                ----------

                                                [ADP][Pi]

It is in contrast with the energy charge, depends on the concentration of Pi and is directly related to
the free energy available from ATP.

Macromolecular complexes
Macromolecular Complexes

With proper positioning of the individual enzymes, the product of one enzymatic reaction can be
passed on directly to the enzyme catalyzing the next step in the metabolic sequence without
equilibrating with the medium.

Significance of chemical equilibria in the regulation of metabolism

Some metabolic steps are so rapid, because of the high activities of the enzymes involved, that
near equilibrium always exists between the starting materials and products of the reactions.
Various dissociable coenzymes such as CoA, FAD, PP, B12, and AMP form common links in other
metabolic processes, consequently, the ratios of free and group charged forms of those coenzymes
can affect the equilibrium of the reactions in which they are involved as well as the metabolic pool
they interconnect. This is the basis of the adenylate control or energy charge hypothesis.

Role of Inorganic Ions in Regulation

Monovalent Cations: Many regulatory enzymes are either dependent upon or are affected by
specific inorganic ions. The ratio of Na+ to K+ can be an important factor in the regulation of certain
cellular activities. K+ activates, pyruvate kinase, aspartokinase, pyruvate carboxylase, δ-ALA
dehydrogenase, and adenosine deaminase. Na+ is generally an antagonist and sometimes quite
inhibitory. But intestinal sucrase is activated by Na+ and is inhibited by K+.

Divalent cations: They have various functions such as activating an enzyme, and carrying electrons
sometime in the binding of the substrate to the enzyme. They also play a role in determining the
secondary, tertiary, and quaternary structure of regulatory enzymes.

Corse control of metabolism

All pathways cannot run at full speed at the same time. If a metabolic pathway is proceeding in one
direction the reaction in the reverse direction must be switched off. In a single pathway such as
glycolysis, its required rate will vary enormously according to the energy needs at the time. The
BMR also vary about 15 times greater during exercise than the resting value.

The fine control mechanism allows sensitive adjustment of flux of nutrients along the metabolic
pathway to the need of the cells under constant environmental conditions. But this may not be
applicable in a severe change. A severe change may arise in higher organisms with a change in diet
or when, in response to other stimuli, the hormonal balance is altered. In starvation, there is a
need to maintain blood glucose level. Live may have to focus on glucose synthesis from non-
carbohydrate precursors. Under this circumstance, enzymes of gluconeogenesis increase and
enzymes of fatty acid synthesis and glycolysis may decrease.

Which of the following is activated by sodium chloride?

Sucrase