1
BI25M1
METABOLIC
REGULATION
LECTURE 1
AIM: To review:
how balance and economy are achieved by
metabolic regulation;
how some metabolic regulation
control over [enzyme].
involves
[Lehninger Edition 4 pp. 21-22; 225-233; Chapter 15
Lehninger Edition 5 pp. 20; 220-227; Chapter 15
Instant Notes
Sections C5, J7]
2
Metabolism
is the flow of energy (and matter)
from the environment to the organism,
through the organism,
and from the organism back to the
environment.
The flow occurs through pathways of
enzyme-catalysed reactions.
[Energy Transformations Lecture 2]
By and large,
organisms remain fairly constant in
composition,
from whole-body to molecular levels,
through long periods of their life.
This stability
(called ‘homeostasis’ by biologists
and ‘dynamic steady state’ by physicists/chemists)
doesn’t mean that nothing is happening.
3
Energy and matter
are continually flowing
(as noted above),
and molecules and cells
are continually lost and replaced
(i.e. are ‘turned-over’).
[Energy Transformations Lecture 2]
So, although outwardly more-or-less
unchanging,
organisms, in fact,
are seething with chemical reactions.
How is such stability achieved?
Answer:
any change in one part of the metabolic
flow system must
1 be detected;
2 trigger a counter-balancing flow change
elsewhere in the flow system.
4
A simple example (at the whole-body level):
in an organism exhibiting homeostasis,
the rate of energy and matter flow INTO
the organism
is balanced
by the rate of flow OUT OF the organism.
If the rate of flow IN changes
(e.g. increases),
this is detected,
and triggers a counterbalancing
(i.e. increased)
change in the flow OUT,
5
so that no net change occurs in the
composition of the organism itself.
So, metabolism is
regulated to achieve balance.
[In growing cells and organisms,
the flow IN is greater than the flow OUT,
and in ill and dying cells and organisms,
the flow IN is less than the flow OUT.]
As well as balance,
metabolic regulation achieves economy.
Particular numbers of cells and molecules
are needed by an organism at any time.
Production of more would be uneconomic,
and is prevented by metabolic regulation.
6
An example (at the molecular level):
A fairly constant [isoleucine] is needed in a
protein-synthesising cell.
At this concentration,
isoleucine acts as an inhibitor of the first
reaction in the metabolic pathway leading
to its own synthesis.
threonine
isoleucine
This ‘feed-back inhibition’
[BI1505 Enzymes lecture
BI25M1 Enzymes Lecture 4]
means that the required [isoleucine] is
maintained in the cell.
Notice how [isoleucine]
is involved in both
the detection system and
the triggering system
mentioned earlier.
7
So, as Lehninger (third edition, p.12) says:
“Living cells are self-regulating chemical
engines, continually adjusting for maximum
economy.”
8
REGULATION
OF
METABOLISM
OCCURS
MAINLY
THROUGH
CHANGES IN ENZYMES
Although [substrates],
[products],
[co-reactants]
pH,
temperature
and other factors
may affect rates of enzyme-catalysed
reactions,
[BI25M1 Enzymes lectures;
BI25M1 Practical 1]
it is through changes in the enzymes
themselves that changes in rates of
metabolic reactions are mainly brought
about.
There are two ways in which this occurs:
1 changes in concentrations of enzymes;
2 changes in activities of enzymes.
9
CHANGES IN METABOLIC REACTION
RATES
THROUGH
CHANGES
IN
ENZYME CONCENTRATIONS
The rate of an enzyme-catalysed reaction
is proportional to [enzyme],
at least, under particular conditions.
Thus, in the presence of a fixed, high
[substrate],
an increase in [enzyme] should increase the
reaction rate,
while a decrease in [enzyme] should
decrease it.
So, in some circumstances,
changes in flow rates through metabolic
pathways might be brought about by
changes in [enzymes].
10
In fact,
the two main ways in which changes in
enzyme concentration are brought about
are:
1 changes in expression of the gene
encoding the enzyme;
2 zymogen activation.
11
CHANGES IN ENZYME CONCENTRATION
BROUGHT ABOUT BY CHANGES IN GENE
EXPRESSION
Changes in [enzyme] occur just as do changes
in the concentration of any biomolecule.
Thus,
an increase in the rate of synthesis
or a decrease in the rate of degradation
of a biomolecule
leads to an increase in [the biomolecule],
while
a decrease in the rate of its synthesis
or an increase in the rate of its degradation
leads to a decrease in its concentration.
In practise,
changes in rates of synthesis are particularly
influential.
12
Enzymes are proteins,
and the main point at which control over the
rate of synthesis of proteins is exerted
is transcription.
The switching on of the lac operon in E. coli
[BI20M3 Transcription lectures]
is an example of
a change in the rate of flow through a
metabolic pathway
that is caused by a change of [enzyme],
which itself is caused by a change in the rate of
enzyme synthesis
produced by a change in the rate of
transcription of the gene encoding the enzyme.
13
increased [lactose] outside E. coli cell
lactose enters cell
allolactose formed
repressor of lac operon deactivated by allolactose
transcription of lac operon unlocked
increased rate of synthesis of mRNA encoding
-galactosidase (transcription)
increased rate of -galactosidase synthesis
(translation)
increased [-galactosidase]
increased rate of lactose catabolism
14
So, the increase in [lactose]
is detected,
and triggers an increase in flow
through the lactose catabolism pathway.
A look back at the process:
a change in [-galactosidase]
is caused by a change in the rate at which it
is synthesised,
which itself is produced by a change in the
rate of transcription of the -galactosidase
gene.
This is an effective, but coarse form of
regulation, because:
1 it takes time for it to begin to operate
(mRNA needs to be made and then
translated);
15
2 when increased flow is no longer
needed, the system takes time to stop
(mRNA and protein need to be
degraded);
3 it is really an on/off switch rather than a
fine-tuning control
(in the absence of lactose, there is no
flow;
the presence of lactose switches on the
flow).
This type of regulation is, therefore,
a way of responding to a fairly long-term
change in the environment of the organism
by producing a fairly long-term change in
the pattern of its intracellular metabolism.
16
CHANGES IN ENZYME CONCENTRATION
BROUGHT
ABOUT
BY
ZYMOGEN
ACTIVATION
Zymogens are inactive precursors that, when
cleaved, become active enzymes.
[Examples include enzymes used in protein digestion
How Organisms Handle Nitrogen Lecture 2]
A particular example:
dietary protein enters stomach
stomach cells stimulated to secrete the hormone gastrin
stomach cells stimulated to secrete pepsinogen
pepsinogen cleaved to pepsin
(autocatalytically, and, later, by pepsin itself)
dietary protein cleaved by pepsin
17
So, the increase in [protein]
is detected,
and triggers an increase in flow
through the protein catabolism pathway.
A look back at the process:
a change in [pepsin]
is caused by a change in the rate at which it
is made from its pre-formed, inactive
precursor.
This is an effective, but still rather coarse
form of regulation:
1 it doesn’t require mRNA synthesis or
translation
(a store of zymogen is ready for
activation when needed);
18
2 once activation begins, further activation
is encouraged
(as pepsin generated acts on remaining
pepsinogen),
so a ‘snowballing’ effect occurs;
3 however, the regulation
is really an on/off switch
(in the absence of dietary protein, there
is no flow;
the presence of protein switches on the
flow).
This type of regulation, like the last, is
a way of responding to a fairly long-term
change in the environment of the organism
by producing a fairly long-term change in
the pattern of its metabolism.
19
Finer forms of regulation,
working inside cells,
rather than changing
flow rates through metabolic pathways by
changing concentrations of enzymes
(e.g. by changes in gene expression
or zymogen activation),
instead
change flow rates by changing
the activities of enzymes already present.
20
BI25M1
METABOLIC
REGULATION
LECTURE 2
AIM: To review:
metabolic regulation involving changes in
enzyme activity;
roles of covalent modification and allosteric
effectors in changing enzyme activity.
[Lehninger Edition 4 pp. 21-22; 225-233; Chapter 15
Lehninger Edition 5 pp. 20; 220-227; Chapter 15
Instant Notes
Sections C5, J7]
21
In Lecture 1, we saw that
changes in flow rates through metabolic
pathways
could be brought about by
changing the concentration of enzymes
(through changes in gene expression or
zymogen activation).
This results in
on/off switches;
rather long-term changes in metabolic
flow rates;
and hence effective, but coarse systems of
metabolic regulation.
A finer form of regulation
involves
not synthesising or degrading the enzymes of
a metabolic pathway faster or slower,
but simply taking the enzyme molecules
already present in the cell
and making them work better (or worse) as
catalysts.
22
This is done by making enzymes interact
with something that affects their activity.
It is potentially a finer form of regulation
because it should:
1 be fast;
2 produce reversible changes
(if the effect of whatever interacts with
the enzyme is reversible);
3 allow fine tuning of metabolic flow rates,
rather than just switching flow on or off
(if changes in enzyme activity can be
made to occur to various degrees).
The two main ways in which changes in
enzyme activity are brought about are:
1 covalent modifications;
2 allosteric effects.
23
CHANGES IN ENZYME ACTIVITY
BROUGHT ABOUT BY COVALENT
MODIFICATION
[Enzymes Lecture 4]
By changing one amino-acid in a
polypeptide,
a base-change (point) mutation
can greatly alter the 3D structure of a
protein,
and hence alter its activity.
An example is the glutamate to valine
change in sickle-cell haemoglobin.
[BI20M1 Genetic Code and Translation Lecture 1]
Similarly,
a temporary (rather than genetic) change in
one amino-acid R group of an enzyme
protein,
should have the potential to alter the 3D
structure of the enzyme,
and hence alter its activity.
24
In fact, several kinds of
reversible,
covalent modifications of R groups of
amino-acid monomers of enzyme proteins
occur inside cells.
Most common is phosphorylation of -OH
groups.
These are present in the R groups of three
coded amino-acids:
serine
threonine
tyrosine.
In some enzymes,
one or more of such amino-acid monomers
can be phosphorylated,
to produce
phosphoserine
phosphothreonine
phosphotyrosine.
25
Phosphorylation changes the amino-acid
monomer R group markedly,
making it bulky and charged.
[This is analogous to the marked structural change
from the R group of glutamate to that of valine in
sickle-cell haemoglobin.]
The change alters the 3D structure of the
enzyme, and hence its activity.
[Similarly, the genetically-caused R group change
alters the activity of the haemoglobin.]
26
The R group phosphorylations
catalysed by kinases,
and are reversed
phosphatases.
by
the
action
are
of
[You have met many other kinases and phosphatases
catalysing similar reactions in BI25M1.]
ATP
ADP
enzyme-OH
enzyme-OP
kinase
Pi
enzyme-OH
enzyme-OP
phosphatase
27
For some enzymes,
phosphorylation activates the enzyme;
for others,
it inhibits the enzyme.
[Examples of both situations are in Lecture 3]
A look back at the process:
a change in enzyme activity
is caused by a change in the 3D structure of
the (already present) enzyme protein
which itself is produced by covalent
modification of an amino-acid R group.
This is an effective, fine form of regulation,
because:
1 it is fast
(as it relies on enzyme-catalysed
changes in protein structure);
28
2 it is reversible
(through the co-ordinated activities of
kinase and phosphatase);
3 it has the potential to allow fine tuning
of metabolic flow rates,
rather than just acting as an on/off
switch
(as phosphorylation and
dephosphorylation can be made to occur
to various degrees).
This type of regulation is, therefore,
a way of responding fast to changing
circumstances.
In a way, it resembles zymogen activation.
There, as here,
a pre-formed biomolecule is made to
change its activity,
but here, the change is not necessarily allor-nothing,
and it is reversible.
29
CHANGES IN ENZYME ACTIVITY
BROUGHT ABOUT BY ALLOSTERIC
EFFECTS
[Enzymes Lecture 4]
Enzymes that are subject to allosteric
effects usually
1
have quaternary structure;
2
show sigmoidal rather than hyperbolic
(Michaelis-Menten) curves when Vo is
plotted against [S].
30
The curve shows that an increase in [S] at
low [S],
e.g. between a and b,
produces a small increase in Vo,
while the same increase at higher [S],
e.g. between b and c,
produces a greater increase in Vo.
This suggests that the presence of low [S]
somehow sensitises the enzyme,
so that it responds to the increase in [S]
at high [S]
more efficiently.
We saw two models
[Enzymes Lecture 4: Concerted and Sequential
models]
that tried to explain the odd, sigmoidal
curve.
31
Both models used the point
allosterically
affected
enzymes
quaternary structure
that
had
to explain that binding of some substrate
increased the efficiency with which the
enzyme
bound more substrate.
[See Enzymes Lecture 4 for details of the models]
32
As well as being sensitive to changes in [S],
the activity of allosterically affected
enzymes is also influenced by other
effectors.
These allosteric effectors bind to a site on
the enzyme other than the active site.
[‘allo steric’ = ‘other shape/structure’]
The binding is non-covalent.
The binding either
stabilises a particular 3D structure of the
enzyme
or
causes the enzyme to a adopt a new,
particular 3D structure.
[The Concerted and Sequential models mentioned
earlier explain what happens in these two different
ways; see Enzymes Lecture 4.]
33
Whatever,
the stabilised or adopted 3D form of the
enzyme
binds substrate differently.
For allosteric activators,
binding to the enzyme
stabilises or induces adoption of a 3D form
that binds substrate better,
and hence the enzyme is activated.
For allosteric inhibitors,
binding to the enzyme
stabilises or induces adoption of a 3D form
that binds substrate worse,
and hence the enzyme is inhibited.
34
Ordinary cell metabolites,
e.g. G6P, ATP,
at certain concentrations,
become allosteric effectors for particular
enzymes.
Thus, in the feed-back inhibition example,
[Lecture 1]
at a particular concentration,
isoleucine becomes an allosteric inhibitor of
the first enzyme of its anabolic pathway.
[Other examples of allosteric effectors are in Lecture
3]
35
A look back at the process:
a change in enzyme activity
is caused by a stabilisation of (or change in)
the 3D structure of the (already present)
enzyme protein
which itself is produced by non-covalent
binding of an effector to an enzyme site
other than the active site.
This is an effective, fine form of regulation,
because:
1 it is fast
(as it relies on binding of an effector to
an enzyme);
2 it is reversible
(as the binding is non-covalent);
36
3 it has the potential to allow fine tuning
of metabolic flow rates,
rather than just acting as an on/off
switch
(as the activation/inhibition can be made
to occur to various degrees).
As we have seen,
allosteric effectors
metabolites.
are
ordinary
cell
Small changes in metabolic flow patterns
inside cells
are detected
as small increases in concentrations of these
metabolites.
At these concentrations,
the metabolites then trigger
counter-balancing flow changes elsewhere
in the flow system
by acting as allosteric activators or
inhibitors.
37
The allosteric effector system, then,
with its subtle detection and triggering
processes,
is a major factor in the maintenance
of the delicate balance and economy
of metabolism inside cells.
38
BI25M1
METABOLIC
REGULATION
LECTURE 3
AIM: To review:
the control of glycogen metabolism,
a process that illustrates several of the
general points made in Lectures 1 and 2
[Lehninger Edition 4 pp. 21-22; 225-233; Chapter 15
Lehninger Edition 5 pp. 20; 220-227; Chapter 15
Instant Notes
Sections C5, J7]
39
Glycogen metabolism is regulated
by a system involving both
covalent modifications and
allosteric effects.
Glycogen is a storage polysaccharide
of skeletal muscle and liver.
[Carbohydrates
Lectures 1, 2)
and
Intermediary
Metabolism
It has different functions in these two sites.
glycogen
catabolism
skeletal
muscle
G6P
liver
G6P
glycolysis
ATP
(for contraction)
Glc
(to replenish blood glucose)
40
Glycogen synthase is a major enzyme of
glycogen anabolism;
glycogen phosphorylase is a major enzyme
of glycogen catabolism.
Both enzymes can be covalently modified by
phosphorylation/dephosphorylation of
serine R groups.
[as discussed in Lecture 2]
41
ATP
ADP
glycogen
phosphorylase b
glycogen
phosphorylase a
phosphorylase kinase*
normally inactive
active
Pi
phosphatase
*We see later that THIS enzyme
can also exist in phosphorylated (active)
and dephosphorylated (inactive) forms.
42
ATP
ADP
glycogen
synthase a
glycogen
synthase b
protein kinase A
active
normally inactive
Pi
phosphatase
43
The role of covalent modifications and
allosteric effects in regulating glycogen
metabolism can be illustrated by
considering events in skeletal muscle
when at rest
during mild exercise
when preparing for vigorous exercise.
When at rest
The non-phosphorylated forms of the two
enzymes predominate.
glycogen phosphorylase b
(normally inactive)
glycogen synthase
(active)
a
Result: at rest, the muscle gradually stores
Glc as glycogen.
44
During mild exercise
Although glycogen phosphorylase b is
normally inactive,
its activity can be altered by allosteric
effectors.
ATP and G6P are allosteric inhibitors of
glycogen phosphorylase b,
and AMP is an activator.
When the muscle is at rest,
the balance of concentrations of the three
effectors keeps
glycogen phosphorylase b inactive.
[as we’ve seen]
45
When the muscle is mildly exercised,
[AMP] rises
[ATP] and [G6P] fall
and glycogen phosphorylase b
is activated.
Result: some glycogen is degraded
to provide ATP for contraction.
46
When preparing for vigorous exercise
With impending ‘fight or flight’,
the hormone epinephrine
is released by the adrenal medulla.
epinephrine
binds to -adrenergic receptor
on membrane of muscle cell
causes GTP to bind to membrane protein Gs
Gs-GTP binds to and activates
membrane adenylyl cyclase
adenylyl cyclase catalyses
ATP
cAMP
[‘cAMP’ is cyclic AMP: its structure is in
Lehninger, Edition 4 p.302; Edition 5 p.298]
cAMP allosterically activates protein kinase A
47
protein kinase A
phosphorylates
glycogen synthase
(a
b)
i.e. inactivates it
phosphorylates
phosphorylase kinase
i.e activates it
[see earlier asterisk*]
glycogen
anabolism
inhibited
which phosphorylates
glycogen phosphorylase
(b
a)
i.e. activates it
glycogen
catabolism
activated
Result: glycogen is degraded to provide
ATP for the contraction necessary in the
stressful circumstances.
48
Why did such a complex system evolve?
The way in which epinephrine
triggers muscle glycogen catabolism
is typical of many other processes
in which signals arriving at a cell
trigger some change in cell activity
e.g.
responses to other hormones and growth factors;
perception of sight, smell, taste;
control of the cell cycle.
All such biosignalling processes
involve the amplification of a signal
through a cascade of events.
49
In the epinephrine/glycogen example,
the signalling process involves a cascading
sequence,
in which a few molecules of an enzyme
act on many other molecules of another
enzyme
which then act on very many molecules of
yet another enzyme
and so on,
so that, at each step,
the signal is amplified.
In this way,
a few hormone molecules
are able to bring about
the mobilisation
of a very large number
of glycogen molecules.
50
A related biosignalling cascade involves
Ca2+ in the regulation of muscle glycogen
metabolism.
nerve impulse signalling contraction
received by muscle cell
causes release of Ca2+ from sarcoplasmic reticulum
into cytosol
Ca2+ activates the dephosphorylated form of
phosphorylase kinase
glycogen phosphorylase is phosphorylated
(b
a)
i.e. activated
glycogen catabolism
activated
Result: glycogen is degraded to provide
ATP for contraction.
51
Relaxation processes
In Lecture 1,
we saw that, in general terms,
for balance and economy in organisms to
be achieved, any change in one part of a
metabolic flow system must
1 be detected;
2 trigger a counter-balancing flow change
elsewhere in the flow system.
In hormonal and Ca2+ regulation of
glycogen metabolism,
the ‘change’ that needs to be detected
is the energy requirement for muscle
contraction,
and the appropriate flow change
(increased glycogen catabolism)
is triggered through the processes seen.
52
Such regulation can only be effective if
there is a way to shut down the triggered
response once the need for it has passed.
Thus, in our example,
glycogen catabolism must not continue,
once the energy needs that triggered it have
been met.
The triggered response must be ‘relaxed’.
Relaxation processes in
catabolism cascades are:
the
glycogen
1 Ca2+ is pumped back from cytosol to
sarcoplasmic reticulum
2 epinephrine production stops
3 GTP is converted to GDP
53
4 cAMP is converted to AMP
5 phosphatase
[seen earlier]
catalyses dephosphorylation of
phosphorylase kinase
(inactivates)
glycogen phosphorylase
(a
b)
(inactivates)
glycogen synthase
(b
a)
(activates)
Result:
[as seen before]
at rest, the muscle gradually stores Glc as
glycogen.