Professional Documents
Culture Documents
Metabolism
MCD Year 1
Anil Chopra
Contents
Metabolism 1 - Introduction to Protein Structure....................................................1
Metabolism 2 - Energetics and Enzymes....................................................................5
Metabolism 3 - Metabolic Pathways and ATP Production I..................................12
Metabolism 4 -Metabolic Pathways and ATP Production II..................................22
Metabolism 5 - Mitochondria and Oxidative Phosphorylation.............................30
Metabolism 6 - Lipids & Membranes.......................................................................35
Metabolism 7 - Cholesterol........................................................................................44
Metabolism 8 - Membrane Trafficking....................................................................52
Metabolism 9 Integration of Metabolism..............................................................55
0
Metabolism 1 - Introduction to Protein Structure
Anil Chopra
1. Outline the reaction by which amino acids are joined together.
Side chains can be polar of non-polar this is vital to the properties of the protein
Eg.
asparagine - polar
1
The polypeptide chain of a protein rarely forms a disordered structure (random
coil) as proteins generally have functions to fulfil, and these functions rely upon
specificity.
In turn, functionality requires a definite 3D structure or conformation of the
polypeptide chain.
Proteins generally possess a degree of flexibility necessary for function e.g.
muscle fibres
Even after synthesis, (post translation), the starting set of 20 amino acids can be
modified to create novel amino acids, enhancing the capabilities of the protein.
Proline can be modified to produce hydroxyproline e.g. collagen fibres, a major
constituent of skin, cartilage, teeth & bones.
These additional hydroxyl groups help to stabilise the fibres.
The addition of sugar residues to the asparagine residues of proteins (N-linked
glycosylation) increases their solubility and also protects them from enzymatic
degradation.
Deficiency of N-linked sugar chain transfer is detected in congenital
carbohydrate-deficient glycoprotein (CGD) syndrome which affects multiple
tissues and has life threatening complications.
Similarly, g-carboxyglutamate is produced by the carboxylation of glutamate.
The formation of g-carboxyglutamate residues within several proteins of the blood
clotting cascade (e.g. factor IX) is critical for their normal function by increasing
their calcium binding capabilities.
The anticoagulant warfarin works by inhibiting the carboxylation reaction.
Folding of Proteins
2
Primary structure is the linear sequence of amino acids that make up the protein.
Secondary structure is defined as local structural motifs within a protein, e.g. -
helices and -pleated sheets.
Tertiary structure is the arrangement of the secondary structure motifs into
compact domains.
Quaternary structure is the three dimensional structure of a multimeric protein
composed of several subunits
5. Distinguish between a -helix and a -pleated sheet and appreciate the bonds
that stabilise their formation.
The helix
Hydrogen Bonds between the C=O of one residue and the N-H of another residue,
4 amino acids along the helix, stabilise the entire structure.
The side chains of individual amino acids project out from within the a helix.
Although theoretically helices can be either right-handed or left handed, the usage
of L-amino acids in proteins means that right-handed helices are favoured.
In proline, the last atom of the side chain is bonded to the main chain N atom.
This prevents the N atom from hydrogen bonding with the C=O groups of another
residue within the helix, thereby distorting the helical conformation, putting a
kink into it.
As with the alpha helix, hydrogen bonds between the N-H and C=O groups of two
or more b-strands hold the b -pleated sheet sheet together.
3
In the b-pleated sheet, the NH and C=O groups point out at right angles to the line
of the backbone. This almost two dimensional sheet is pleated, like the bellows of
an accordion.
Alternate b -strands can run in the same direction to give a parallel b-pleated sheet
or in opposite directions to give an antiparallel b -pleated sheet.
The pleating in each case allows for the best alignment of the hydrogen bonded
groups.
Covalent bonds (in which two atoms share electrons) are the strongest bonds
within protein and exist in the primary structure itself. Covalent bonds can also
exist as disulphide bridges. These occur when cysteine side chains within a
protein are oxidised resulting in a covalent link between the two amino acids.
Hydrogen Bonds occur when two atoms bearing partial negative charges share a
partially positively charged hydrogen, the atoms are engaged in a hydrogen bond
(H-bond).
Ionic interactions arise form the electrostatic attraction between charged side
chains e.g. Glu, Asp, Lys and Arg. They are relatively strong bonds, particularly
when the ion pairs are within the protein interior and excluded from water.
Van der Waals Forces are transient, weak electrostatic attractions between two
atoms, due to the fluctuating electron cloud surrounding each atom which has a
temporary electric dipole. Although relatively weak and transient in nature,
because of the sheer number of these interactions within a protein, they can still
have a large part to say in the overall conformation of a protein.
Hydrophobic Interactions are a major force driving the folding of proteins into
their correct conformation. They juxtapose hydrophobic side chains by packing
them into the interior of the protein. This creates a hydrophobic core and a
hydrophilic surface to the majority of proteins
Summary
Proteins are chains of amino acids linked by peptide bonds which have evolved to
fulfil specific functions within the cell.
Such functions are reliant upon the 3D structure or conformation of the protein
which is held together by a variety of forces.
The a-helix and b-pleated sheet are the two staple motifs that define the
conformation of a protein.
The nature of the amino acid side chain dictates its position within the
conformation of the protein.
Post-translational modifications of proteins add yet more diversity to protein
structure.
4
Metabolism 2 - Energetics and Enzymes
Anil Chopra
Energy can neither be created nor destroyed. i.e. it is simply converted from one
form to another.
In any isolated system, e.g. a single cell or the universe, the degree of disorder
can only increase.
The amount of disorder in a particular system can be quantified as its entropy.
Reactions proceed spontaneously towards products with greater entropy (i.e. more
disorder).
2. Explain the concept of free energy and how we can use changes in free energy
to predict the outcome of a reaction.
However, biological systems are very well ordered.
This is achieved by investing taking energy from the environment surrounding the
cell and investing it in chemical reactions which maintain order.
Increased
disorder
cell Increased
order
HEAT
Surrounding environment
5
(Gibbs) Free Energy is defined as the amount of energy within a molecule that
could perform useful work at a constant temperature.
It is denoted by the letter G and has units of kilojoules/moles (kJ/mole).
The free energy function combines both the 1st and 2nd Laws of thermodynamics.
Changes in G (DG) measure the amount of disorder that results from a particular
reaction.
i.e. In the above scenario, DG measures both the change in order within the cell
and also upon the change in entropy of the system.
Lets consider the reaction:
A+B C+D
reactants products
The changes in free energy for this reaction (DG) can be defined by:
DG = free energy (C+D) - free energy (A+B)
A reaction can only occur spontaneously if DG is negative.
Conversely, a reaction cannot occur spontaneously if DG for the reaction is
positive.
3. Draw the chemical structure of ATP and explain how it acts as a carrier of free
energy and is used to couple energetically unfavourable reactions.
6
In a biological setting, most energetically favourable reactions will not occur at a
rate useful for life, unless catalysed by enzymes. Enzymes function by lowering
the barriers that block a particular reaction.
Enzymes bind one or more substrate molecules tightly within a part of the protein
known as the active site. Enzymes arrange the substrate(s) in such a way that
certain bonds are strained.
Key residues within the enzyme participate in either the making or breaking of
bonds by altering the arrangement of electrons within the substrate(s).
This can often take the form of either oxidation reactions, (in which electrons are
removed from a molecule) or reduction reactions (in which electrons are added
to a molecule).
Since the cellular environment is generally aqueous, often, when a molecule gains
an electron, it also simultaneously gains a proton.
The transition state is the particular conformation of the substrate in which the
atoms of the molecule are rearranged both geometrically and electronically so that
the reaction can proceed.
Enzymes work by bending their substrates in such a way that the bonds to be
broken are stressed and the substrate molecule resembles the transition state. This
makes them more amenable to reaction with other molecules.
Enzymes function by lowering the barriers that block a particular reaction. Put
graphically:
Transition State
of Substrate Transition State
of Substrate
Substrate
Free energy
Substrate Activation Energy Activation Energy
Product Product
reaction
Lysozome
Lysozyme is a component of tears and nasal secretions and is one of the first lines
of defence against bacteria.
It catalyses the hydrolysis of sugar molecules within bacterial cell walls that are
necessary for their structure. With this bond broken, the bacteria lyse and die.
The activity of lysozyme was discovered by Sir Alexander Fleming, who suffering
from a cold, allowed some of his nasal secretions to drip into a bacterial culture.
This results in lysed bacteria.
Lysozyme hydrolyzes alternating polysaccharide copolymers of N-acetyl
glucosamine (NAG) and N-acetyl muramic acid (NAM) which represent the
"unit" polysaccharide structure of many bacterial cell walls.
Lysozyme cleaves at the b(1-4) glycosidic linkage, connecting the C1 carbon of
NAM to the C4 carbon of NAG.
7
Glu35 protonates the oxygen in the glycosidic bond breaking the bond holding the
two sugar molecules together.
A water molecule enters and is de-protonated by Glu35.
Asp52 stabilises the positive charge in the transition state.
The hydroxide ion attacks the remaining sugar molecule adding an OH group.
Both Glu35 and Asp52 are in their original state to continue catalysis.
Glu35 protonates the oxygen in the glycosidic bond breaking the bond holding the
two sugar molecules together.
A water molecule enters and is de-protonated by Glu35.
Asp52 stabilises the positive charge in the transition state.
The hydroxide ion attacks the remaining sugar molecule adding an OH group.
Both Glu35 and Asp52 are in their original state to continue catalysis.
Glucose-6-Phosphatase
H2O
+ Pi
Glucose-6-phosphatase
Glucose-6-phosphate Glucose
Glucose-6-Phosphatase Deficiency
6. Outline the differences between lock and key and induced fit models of
substrate-enzyme interactions.
8
Lock and Key Model
+
enzyme substrate enzyme-substrate
complex
In this model, the shape of the substrate (key) matches that of the active site (lock)
of the enzyme.
This model explains the specificity of most enzymes for a single substrate.
In this model, the substrate induces a change in the conformation of the enzyme
which results in the formation of the active site. Upon release of products, the
enzyme reverts back to its original conformation.
+
enzyme substrate enzyme-substrate
complex
Substrate Concentration
Vmax
Vmax
rate of reaction
substrate concentration
Km
9
The reaction rates of enzymes vary considerably and can be measured
experimentally. This is useful if we are testing an enyme inhibitor e.g. captopril
Km is known as the Michaelis Constant ands is defined as the concentration of
substrate at which a particular enzyme works at half its maximal velocity.
Biochemically, the Km value is useful as a means of comparing the strength of
Enzyme-Substrate complexes.
Generally a low Km indicates tight binding of a substrate to an enzyme.
Conversely, a high Km is indicative of weak binding.
Temperature
rate of reaction
temperature
pH Level
rate of reaction
pH
10
NAD+ (Nicotinamide
adenine dinucleotide) is a
vital component of many
dehydrogenation reactions
within the body.
It can be described as a co-
enzyme as it has no
catalytic activity of its own
and functions only after
binding to a enzyme.
NAD+ catalyses the dehydrogenation of substrates by readily accepting a
hydrogen atom and two electrons.
+ H+ + 2e-
H- (hydride ion)
NAD+ NADH
Lactate Dehydrogenase
Malate Dehdrogenase
11
Metabolism 3 - Metabolic Pathways and ATP Production
I
Anil Chopra
1. Sketch a cartoon of the three stages of cellular metabolism that convert food to
waste products in higher organisms, illustrating the cellular location of each
stage.
Metabolism
12
Glucose
Free
Ener
gy
Free energy
Relatively large
released as heat
activation energy
overcome by heat
source CO2
+ H20
Reaction
Free
Energy
CO2
Relatively small activation energies
overcome by enzymes and body
+ H20
temperature
Reaction
Reaction
This is around 50% efficient, c.f. car engines which on
average are only 20% efficient
13
2. Outline the metabolism of glucose by the process of glycolysis, listing the key
reactions, in particular those reactions that consume ATP and those that
generate ATP.
Overview of Glycolysis
1x 6C glucose 2x 3C pyruvate 2x ATP produced
Essentially an anaerobic process
Occurs in cytoplasm of cells
Ten reactions that make up glycolysis pathway can be split into two main
concepts:
Glycolytic Pathway
hexokinase
1. glucose glucose-6-phosphate
ATP ADP
phosphoglucose isomerase
2. glucose-6-phostphate fructose-6-phosphate
phosphofructokinase
3. fructose-6-phosphate fructose-1,6-
bisphosphate
ATP ADP
adolase
4. fructose-1,6-bisphosphate glyceraldehyde-3-phosphate
dihydroxyacetone phosphate
14
- opening of the fructose ring to generate two high energy compounds, one
of which, (dihydroxyacetone phosphate) subsequently undergoes
isomerisation.
At this half-way point in the pathway one mole of glucose has given rise to two
moles of glyceraldehyde-3-phosphate.
So far, no energy has yet been produced but two moles of ATP have been used.
glyceraldehyde
3-phosphate dehydrogenase
6. 2x glyceraldehyde-3-phosphate 2x 1,3-
bisphosphoglycerate
NAD+ + Pi NADH
- NADH is generated here which can be later used to generate yet more ATP
within the mitochondria in a process known as oxidative phosphorylation.
phosphoglycerate kinase
7. 2x 1,3-bisphosphoglycerate 2x 3-phosphoglycerate
ADP ATP
phosphoglycerate mutase
8. 2x 3-phosphoglycerate 2x 2-phosphoglycerate
enolase
9. 2x 2-phosphoglycerate 2x phosphoenolpyruvate
dehydration
pyruvate kinase
15
ADP ATP
- transfer of the high energy phosphate group to ADP, generating one ATP
molecule in the process.
There is are 2 molecules of pyruvate produced and a net gain of 2 ATP per
molecule of glucose commited to the glycolytic pathway.
phosphoglycerate kinase
ADP ATP
Alcoholic fermentation:
. pyruvate decarboxylase
pyruvate acetaldehyde
H+ CO2
alcohol dehydrogenase
acetaldehyde ethanol
NADH + H+ NAD+
Generation of lactate:
lactate dehydrogenase
16
pyruvate lactate
NADH + H+ NAD+
Regeneration of NAD+
Both alcoholic fermentation and the generation of lactate serve one common
purpose:
They allow NAD+ to be regenerated and thus glycolysis to continue, in conditions
of oxygen deprivation. i.e. conditions in which the rate of NADH formation by
glycolysis is greater than its rate of oxidation by the respiratory chain.
- stroke
- heart attack
- liver disease (eg. hepatitis)
- muscle injury
- muscular dystrophy
- pulmonary infarction
Creatine kinase
17
ADP + H+ ATP
When a muscle is damaged, creatine kinase leaks into the bloodstream. Either
total levels of creatine kinase or the tissue specific isoform can be measured to
help to determine which tissue has been damaged.
Elevated levels can be used to:
The total creatine kinase test is about 70% accurate whilst isoenzyme testing is
about 90% accurate.
NAD+ NADH
18
Lipoamide
The long flexible arm of the molecule allows the dithiol group to swing from one
active site to another within the complex.
Arsenite (AsO33-) and mercury have a high affinity for neighbouring sulphydryl
groups, such as those that occur in reduced lipoamide and will readily inhibit
pyruvate dehydrogenase.
FAD + 2 e- + 2 H+ FADH2
6. Describe the different processes by which the fatty acid palmitate and the
amino acid alanine are converted into acetyl-CoA.
Acetyl CoA is produced from both types of major food molecules within the
mitochondria of cells.
Thus it is the location where most of the cell's oxidation reactions occur and also
where the majority of the cells ATP is made.
19
Fatty Acid Metabolism
By virtue of being fully reduced (i.e carbon skeletons 'saturated' with hydrogens)
the oxidation of fatty acids constitutes the most compact fuel for the body's energy
requirements and as a result, fatty acid oxidation yields several times the usable
chemical energy that carbohydrates can deliver.
On a weight basis, the caloric yield from fatty acids is about double that from
carbohydrates. More than half of the body's energy needs including the liver, but
not the brain, comes from fatty acid oxidation and this is enhanced during fasting
over long periods of time.
Fatty Acids are metabolised in the mitochondria in several stages. Firstly, they are
converted into an acyl CoA species:
Amino acid metabolism can be 'separated' into pathways depending on the number
of carbon atoms the amino acid possesses. All of the degradation pathways
produce common end products which can enter the TCA. The majority of the
degradation takes place in the liver.
C3 family e.g. alanine, serine (glycine), and cysteine are all degraded to pyruvate.
C4 family e.g. aspartate and asparagine are degraded to oxaloacetate.
C5 family e.g. glutamine, proline, arginine, and histidine, all of which are
converted to a-ketoglutarate.
Protein Metabolism
20
Alanine (C3) undergoes transamination by the action of the enzyme alanine
aminotransferase.
Summary
21
Metabolism 4 -Metabolic Pathways and ATP Production
II
Anil Chopra
1. Outline the Krebs or TCA (tricarboxylic acid cycle) with particular reference to
the steps involved in the oxidation of acetyl Co-A and the formation of NADH
and FADH2 and the cellular location of these reactions.
pyruvate dehydrogenase
complex
22
NAD+
NADH
1. Decarboxylation of pyruvate to
give hydroxyethyl TPP.
2. Oxidation & transfer to
lipoamide to give
acetylipoamide.
3. Transfer of the acetyl group to
CoA to give acetyl CoA.
4. Regeneration of oxidised
lipoamide.
5. Regeneration of oxidised FAD,
generating NADH.
Acetyl CoA can also be manufactured from palmitic acid (a fatty acid).
The -oxidation reactions continue to consecutively remove 2-carbon units from
the acyl CoA therby producing acetyl CoA.
On the final cycle (4-carbon fatty acyl CoA intermediate), two acetyl CoA
molecules are formed.
From just 7 b-oxidation reactions, the 16-carbon palmitoyl CoA molecule
produces 8 molecules of acetyl CoA.
During each cycle one molecule each of FADH2 and NADH are produced. The
overall reaction of -oxidation of palmitoyl CoA is:
a.k.a. The Tricarboxylic Acid (TCA) cycle or The Citric Acid Cycle.
A continuous cycle of eight reactions, starting with 2 carbon atoms from acetyl
CoA being condensed with the 4 carbon unit of oxaloacetate to give a 6 carbon
unit , citrate.
The thio-ester linkage of the acetyl
CoA allows it to be readily donated
to oxaloacetate.
Each turn of the cycle produces two
molecules of CO2 (waste) plus
three molecules of NADH, one
molecule of GTP and one molecule
of FADH2.
23
citrate synthase
Step 1:
Step 2: aconitase
24
Step 6: succinate dehydrogenase
FAD FADH2
H2O
NAD+ NADH + H+
The Krebs cycle enzymes are soluble proteins located in the mitochondrial matrix
space, except for succinate dehydrogenase, which is an integral membrane protein
that is firmly attached to the inner surface of the inner mitochondrial membrane.
Here, it can communicate directly with components in the respiratory chain, as we
shall see in the next lecture.
The majority of the energy that derives from the metabolism of food is generated
when the reduced coenzymes are re-oxidised by the respiratory chain in the
mitochondrial inner membrane in a process known as oxidative phosphorylation.
The Krebs cycle only operates under aerobic conditions, as the NAD+ and FAD
needed are only regenerated via the transfer of electrons to O2 during oxidative
phosphorylation.
ATP production by glycolysis and the Krebs cycle is only a prelude to oxidative
phosphorylation.
The function of the Krebs cycle is to produce the reduced co-factors NADH and
FADH2 which are re-oxidised during osidative phosphorylation to yield the
following:
25
- three ATP molecules formed by the re-oxidation of each NADH
molecule
- two ATP molecules formed by the re-oxidation of each FADH2
molecule.
This takes place primarily in the heart and liver and uses two membrane carriers
and four enzymes.
The net reaction in terms of NADH is:
26
However, oxaloacetate has now been
being depleted from the cytoplasm and is
accumulating within the mitochondrial
matrix.
Since it cannot cross the matrix
membrane, the problem cannot be solved
by by simply transferring oxaloacetate
back to the cytoplasm.
Instead, the cell uses a transamination
reaction to take an amino group from glutamate and transfer it to oxaloacetate,
giving aspartate.
This aspartate then crosses the matrix membrane, via an amino acid transporter,
and is duly converted by the same transamination reaction in reverse, back to
oxaloacetate.
Transamination is therefore pivotal to the malate-aspartate shuttle it is defined as
a reaction in which an amine group is transferred from one amino acid to a keto
acid, thereby forming a new pair of amino and keto acids.
3. Explain in general terms the relationship between TCA intermediates and those
pathways involved in amino acids synthesis and breakdown.
The general strategy of amino acid degradation is to remove the amino group
(which is eventually excreted as urea) whilst the carbon skeleton is either
funnelled into the production of glucose or fed into the Krebs cycle
Degradation of all twenty amino acids gives rise to only seven molecules,
pyruvate, acetyl CoA, acetoacetyl CoA, a-ketoglutarate, succinyl CoA, fumarate
and oxaloacetate.
27
4. Calculate the total yield of ATP obtained from the complete oxidation of one
glucose molecule.
glycolysis
PDH
TCA cycle
This gives 2x ATP + 10x NADH (3x ATP each) + 2x FADH2 (2x ATP each) + 2x GTP
= 38x ATP from one molecule of glucose
5. Compare the complete oxidation of one glucose molecule, with the beta oxidation
of palmitic acid with reference to the ATP produced per molecule of each
substrate.
1st step
palmitate + ATP + HS-CoA palmitoyl CoA + AMP + PPi
(loss 2xATP)
-oxidation
TCA cycle
This gives 31xNADH (3x ATP each) + 15x FADH2 (2xATP each) + 8x GTP 2xATP
= 129x ATP from one molecule of plamitate about 5 times that from glucose
Glycolysis and the Krebs cycle provide the starting point for many biosynthetic
reactions.
The amino acids, nucleotides, lipids,
sugars, and other molecules shown here
as products, in turn, become the
precursors for the many of the
macromolecules of the cell.
28
Each black arrow in this diagram denotes a single enzyme-catalysed reaction. Red
arrows generally represent multi-step pathways.
If Krebs cycle intermediates are drawn off for biosynthesis then they must be
replenished, otherwise the cycle will grind to a halt. e.g. If oxaloacetate is
removed, acetyl CoA cannot enter and glycolysis backs up.
Thankfully some enzymes can catalyse anaplerotic reactions (from the Greek to
fill up) which can regenerate Krebs cycle intermediates.
29
Metabolism 5 - Mitochondria and Oxidative
Phosphorylation
1. Outline the proposed evolutionary origins of mitochondria.
The electron transport chain is a chain of three complexes and two mobile carriers
whih act as electron carriers.
Membrane complexes
- NADH dehydrogenase complex
- cytochrome b-c1 complex
- cytochrome oxidase complex
Mobile carriers
- ubiquinone (co-enzyme Q)
- cytochrome C
These proteins accept electrons and in doing so, a proton (H+) from the aqueous
solution.
As electrons pass through each of the complexes, protons are pumped to the
intermembrane space.
Each unit of the chain has a higher affinity for electrons than the previous unit,
allowing them to flow in a logical order.
The transfer of electrons from one complex to another is energetically favourable
and so the electrons lose energy as they progress along the chain.
Ubiquinone
Ubiquinone can pick up either one or two electrons (with an H+ from solution) and
pass them to cytochrome b-c1 complex.
Its hydrophobic tail confines it to the lipid bilayer of the membrane where it is
needed.
It is the entry point for electrons donated by FADH2 since succinate
dehydrogenase can communicate directly with it.
As fewer protons are pumped to the intermembrane space than for NADH, less
ATP is produced.
Cytochrome Oxidase
31
In addition, 4 protons are pumped to the intermembrane space, enhancing the
proton gradient.
Redox Reactions
The reactions that take place in the electron transport chain are redox reactions
since a reduced substrate donates electrons and an oxidised substrate accepts them
(thus electrons move along the transport chain).
The ability of a redox couple to accept of donate electrons is known as the
reduction potential, or redox potential.
Standard redox potential is given the symbol E0.
Positive E0 value shows redox couple has a tendency to donate electrons and vice
versa.
Chemiosmosis:
The proton motive force that drives H+ back into the matrix space consists of a pH
gradient and a transmembrane electrical potential.
This flow of protons back into the matrix is coupled to ATP synthesis.
Protons flow back into the matrix through the ATP synthase molecule, which
produces ATP from ADP + Pi.
ATP Synthase:
32
As the subunit functions as an asymmetrical axle, the subunits are compelled
to undergo structural changes.
This rotation drives the transitions of the catalytic portions of the subunits,
which in turn, alters their affinities for ATP and ADP.
As a consequence, torsional energy flows from the catalytic subunit into the bound
ADP and Pi to promote the formation of ATP.
The direction of proton flow dictates ATP synthesis vs ATP hydrolysis.
5. Explain why carbon monoxide, cyanide, malonate and oliogomycin are poisonous
in terms of their effects on specific componenets of the electron transport chain.
Malonate Poisoning
Malonate closely resembles succinate and acts as a competitive inhibitor of
succinate dehydrogenase.
This Krebs Cycle enzyme resides in the the inner membrance and passes FADH2
directly to ubiquinone.
Malonate therefore slows down the flow of electrons from succinate to
ubiquinone, slowing down ATP production.
Oliogomycin Poisoning
Oliogomycin is an antibiotic that inhibits oxidative phosphorylation by binding
within the stalk of ATP synthase
This blocks the flow of protons through the enzyme.
ATP synthesis is inhibited and a backlog of protons builds up in the
intermembrane space.
This inhibits the flow of electrons through the electron transport chain as the H+
concentration in the intermembrane space reaches saturation point no more can
be pumped out.
33
6. Describe how oxidative phosphorylation can be measured experimentally.
Summary
Mitochondria are present in almost all eukaryotic cells and are believed to be
evolutionary descendants of an earlier prokaryote life form.
The majority of ATP produced within the body occurs a result of oxidative
phosphorylation, a process which takes place within the mitochondria.
In this process, electrons from the reduced co-enzymes NADH and FADH2 are
passed via a series of enzyme complexes, called the electron transport chain,
which ultimately results in the reduction of oxygen to water and the pumping of
protons out of the mitochondrial matrix, to generate a proton gradient.
These protons re-enter the matrix via the molecular turbine ATP synthase, which
couples the resultant kinetic energy to ATP production.
Compounds which inhibit either the electron transport chain or the proton
gradient, disrupt oxidative phosphorylation which be measured experimentally
with an oxygen electrode.
34
Metabolism 6 - Lipids & Membranes
Objectives:
Describe the structure of: fatty acids, triglycerides, phospholipids,
cholesterol and sphingomyelin.
Give examples of how the lipid composition can differ for different
cellular membranes, and indicate the significance of this.
Outline the pathway for synthesis of fatty acids.
Distinguish between the pathways for synthesis and metabolism of
fatty acids in terms of: substrates and products, coenzymes used,
carrier molecules and cellular location.
Fatty acids:
Triglycerides:
35
R1, R2 and R3 indicate the three fatty acids.
Phospholipids:
Cholesterol:
Sphingomyelin:
This is the
composition of
36
sphingomyelin. As can be seen it has two components; phosphocholine
and ceramide.
Give examples of how the lipid composition can differ for different
cellular membranes, and indicate the significance of this:
The degree of fluidity of the cell membrane (the ease with which
its lipid molecules can move about) is important for membrane
function, and thus must be maintained within certain limits.
37
The fluidity of a lipid bilayer at a given temperature depends on its
phospholipids composition, and especially on the nature of the
hydrocarbon tails: the closer and more regular the packing of the
tails, the more viscous the and less fluid the bilayer will be. There
are two major properties of hydrocarbon tails that affect their
packing in the bilayer one is their length, and the other is their
unsaturation (that is the number of double bonds they contain).
Hydrocarbon tails of phospholipids vary in length between 14 and
20 carbon atoms, 18-20 being the most usual. A shorter chain
reduces the tendency of the hydrocarbon tails to interact with one
another, therefore increasing the fluidity of the membrane.
Each double bond in a hydrocarbon tail creates a kink in the tail,
making it harder for the tails to pack closely together. Thus, lipid
bilayers that contain a large proportion of unsaturated
hydrocarbon tails are more fluid than those with lower proportions.
38
AcetylCoA
is the key
39
2nd STEP: ACTIVATION BY ACYL CARRIER PROTEIN
(similar to CoA activation in -oxidation).
3rd STEP: ELONGATION BY SUCCESSIVE ADDITION OF
2-CARBON UNITS (this is catalysed by the enzyme FA
Synthase):
40
Fatty acid synthase mechanism of reaction
41
The overall reaction is given as
Lipogenesis is regulated:
1. Feedback
inhibition of
palmitoyl
CoA to:
A) Acetyl-
CoA
carboxylase.
B) FA
synthase.
C) Pentose
phosphate
pathway.
2. Acetyl
CoA
carboxylase
regulation by
hormones.
42
3. Transcriptional regulation of acetyl CoA carboxylase and FA
synthase (activated by insulin and inhibited by glucagon.
The pathway for the metabolism of fats is given below (the actual
pathway is highlighted in the pale yellow colour)
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Metabolism 7 - Cholesterol
Objectives:
Explain the physiological functions of cholesterol in membrane
stability.
Outline the synthesis of cholesterol from acetate.
Outline the synthesis of bile acids and steroid hormones from
cholesterol.
Describe the mechanism of transport of cholesterol around the
body and its uptake into cells.
Draw a diagram of low density lipoprotein (LDL).
Explain why disturbances in cholesterol homeostasis cause disease.
Give an example of how a selective enzyme inhibitor can be used
as a pharmacological agent in controlling cholesterol metabolism.
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1.1
HMGCoA
Reductase as
the regulated
step in 1.2
cholesterol
synthesis
1.3
C5
C10
C15
C30
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Outline the synthesis of bile acids and steroid hormones from
cholesterol:
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Levels of steroid hormone controlled by the rate of synthesis.
Cholesterol desmolase generates pregnenolone the precursor of all
steroid hormones.
There are 5 classes of steroid hormone:
1. Progestins (progesterone, 17-Hydroxypregnenolone, 17-
Hydroxyprogesterone)
2. Glucocorticoids
3. Mineralocorticoids
4. Androgens
5. Estrogens
Conjugated bile
acids
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This pathway represents the major route for elimination of
cholesterol via the GI tract.
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Draw a diagram of low density lipoprotein (LDL):
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Metabolism 8 - Membrane Trafficking
1. Explain the terms endocytosis and exocytosis.
Exocytosis is the process of secreting macromolecular material from a cell.
It involves the fusion of a membrane-enclosed intracellular vesicle with the
plasma membrance, followed by the opening of the vesicle and the emptying of its
contenets to the outside.
Endocytosis is the mechanism of uptake of macromolecular material into a cell
from the outside.
It typically involves formation of a coated pit on the plasma membrane, which
buds off into the cytoplasm to form a coated vesicle which delivers its contents to
an endosome.
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At the trans golgi network NANA is added and the proteins are sorted.
The golgi apparatus also returns ER resident proteins recognised by KOEL
receptors.
Here proteins are sorted and packaged into vesicle depending on whether they are
to be released by constitutive or regulated secretion.
Lysosomal emnzymes are also sorted from others.
The mannose in these proteins is phosphorylated in the golgi stack producing
mannose-6-phosphate.
This is then recognised by a M6P receptor and these proteins are taken in a
receptor dependent transport vesicle to a lysosome.
The M6P receptors are recycled by budding from a late endosome.
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The LDL binds with an LDL receptor at the plasma membrane and is taken into a
coated vesicle via endocytosis.
The vesicle is uncoated in order to fuse with an early endosome.
The LDL receptors bud off the early endosome and it becomes a late endosome.
With the introduction of hydrolytic enzymes a lysosome is formed and free
cholesterol is released into the cytosol.
At the donor membrane, the cargo is sorted and a vesicle forms and buds off the
plasma membrane.
The vesicle then moves through the cytosol vesicles move along microtubules to
find receptors.
When the vesicle reaches specific receptor molecules on the receptor membrane
recognition occurs between this and specific molecules on the vesicle, resulting in
vesicle tethering or docking to the acceptor membrane.
The vesicle membrane and acceptor membrane then fuse together, opening the
vesicle and releasing the contenets into the lumen of the acceptor organelle.
6. Give examples of diseases resulting from defects in the secretory and endocytic
pathways.
A disease that results from a defect in the secretory pathway is cystic fibrosis.
This results from blocks at the exit of the ER due to misfolding of the proteins.
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Metabolism 9 Integration of Metabolism
1. Distinguish the features of metabolic activity in the following tissues: liver, brain,
muscle, adipose tissue.
Liver
Plays central role in coordinating metabolism throughout the body.
Immediate recipient of nutrients absorbed at the intestines.
Wide repertoire of metabolic processes.
Highly metabolically active and can interconvert nutrient types.
Central role in maintaining blood glucose at 4.0-5.5 mM.
Storage organ (glycogen).
Central role in lipoprotein metabolism.
Brain
Has continuous high ATP requirement, cannot utilise fats.
Requires continuous supply of glucose for metabolism.
Cannot metabolise fatty acids
Ketone bodies (-hydroxy-butyrate) can partially substitute for glucose.
Too little glucose (hypo-glycaemia) causes faintness and coma.
Too much glucose (hyper-glycaemia) can cause irreversible damage.
Muscle
Can have periods of very high ATP requirement during vigorous contraction.
During vigorous contraction ATP consumption is faster than supply by oxidative
phosphorylation (O2 diffusion is limiting).
Energy stores of glycogen (glucose-6-P for glycolysis) and creatine phosphate
(ATP).
Under anaerobic conditions pyruvate is converted to lactate or alanine which can
leave muscle and reach the liver via the blood.
Adipose tissue
Is a long-term storage site for fats.
3. Describe the changes in metabolic activity while eating and while fasting.
On having a meal, blood glucose initially rises and is controlled by:
- Increased secretion of insulin (and reduced glucagon) from islets.
- Increased glucose uptake by liver - used for glycolysis and glycogen synthesis.
Acetyl-CoA produced is used for fatty acid synthesis.
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- Increased glucose uptake and glycogen synthesis in muscle.
- Increased triglyceride synthesis in adipose tissue.
- Increased usage of metabolic intermediates throughout the body due to general
stimulatory effect on synthesis and growth.
After a meal blood glucose starts to fall and is controlled by:
- Increased glucagon secretion (and reduced insulin) from islets.
- Glucose production in liver resulting from gluconeogenesis and glycogen
breakdown.
- Utilisation of fatty acid breakdown as alternative substrate for ATP production.
- [NB adrenaline has similar effects on liver, but also stimulates skeletal muscle
towards glycogen breakdown and glycolysis, and adipose tissue towards fat
lipolysis to provide other tissues with alternative substrate to glucose]
After prolonged fasting (longer than can be covered by glycogen reserves):
- Glucagon/insulin ratio increases further.
- Adipose tissue begins to hydrolyse triglyceride to provide fatty acids for
metabolism.
- TCA cycle intermediates are reduced in amount to provide substrate for
gluconeogenesis.
- Protein breakdown provides amino acid substrates for gluconeogenesis.
- Ketone bodies are produced from fatty acids and amino acids in liver to
substitute partially the brains requirement for glucose.
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5. Describe the metabolic processes during vigorous muscular activity and explain
why acidosis can result.
During vigorous contraction ATP consumption is faster than supply by oxidative
phosphorylation (O2 diffusion is limiting).
Further ATP by interconversion from creatine phosphate.
Glycogen stores provide glucose for anaerobic metabolism only (glycolysis).
Pyruvate is converted to lactate or alanine - otherwise it would build up and the
pathway would be inhibited by excess product.
Lactate/alanine pass into the blood and the liver uses them to replenish glucose by
gluconeogenesis
.
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