Thursday, January 7, 2016
Lecture 1. Energy in Biological Systems I.
- Lodish et al., pp. 43‐45; 49‐51; Berg et al., pp. 427‐429.
- E.Coli is a very simple organism; it grows and divides quickly. It requires requires a
carbon source (Glucose), NH , 4O and 4alts. It must convert all those into amino
acids, nucleotides, lipids, sugars, vitamins (coenzymes) and macromolecules (DNA,
RNA, proteins, polysaccharides). The synthesis and breakdown occurs through
interconnected pathways involving many different reactions called the metabolic
- Cellular reactions are governed by
rules that govern all chemical
reactions. Each reaction is catalyzed
by speciﬁc enzymes binding speciﬁc
- Organisms devote much of their
genome to specifying metabolic
proteins (see table on the right). A lot of
genes have metabolic function.
- Basic principles of a cell’s
1. Fuels are degraded and large
molecules are constructed step by
step in a series of linked reactions
called metabolic pathways.
2. An energy currency common to all
life forms, adenosine triphosphate
(ATP), links energy-releasing
pathways with energy-requiring
3. The oxidation of carbon fuels powers the formation of ATP.
4. Although there are many metabolic pathways, a limited number of types of
reactions and particular intermediates are common to many pathways.
5. Metabolic pathways are highly regulated.
1 Thursday, January 7, 2016
Lecture 2. Energy in Biological Systems II.
- Lodish et al., pp. 51 – 52, 78‐81; Berg et al. pp. 429‐430.
- An enzyme’s active site consists of two functionally important regions: the substrate
binding site, which recognizes and binds the substrate or substrates, and the catalytic
site, which carries out the chemical reaction once the substrate has bound.
- Reactions that release energy are exergonic (release energy) and catabolic
(breaking down). On the other hand, endergonic reactions are anabolic (building
- What is energy? Energy is the ability to do work from a scientiﬁc standpoint.
- Energy can be kinetic (muscle contraction) or
potential ( chemical bonds , concentration
gradients , charge separation across
membranes) and energy can be converted from
one form into another.
- All chemical reactions are reversible. At
equilibrium, the forward and reverse rates are
the same. The equilibrium constant is
characteristic of a reaction.
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- We don’t really deﬁne the Gibb’s free energy
(G), we care about the change in free
energy. According to the picture on the right,
ΔG dictates the side of the reaction:
• if ΔG<0, the forward reaction is favoured
• if ΔG>0, the reverse reaction is favoured
• if ΔG=0, the system is at equilibrium
- An important thermodynamic fact is that the overall free-energy change for a
chemically coupled series of reactions is equal to the sum of the free-energy changes
of the individual steps. In other words, standard free energy values are additive.
Therefore It is possible to form higher energy compounds like glutamine from lower
energy compounds like glutamate and ammonia by coupling their synthesis to energy
yielding (exergonic) reactions like the hydrolysis of ATP.
3 Thursday, January 7, 2016
- The standard free energy change is ΔG . In the equation on the bottom, 2.303 is the
factor to convert the “ln" to “log”.
- The table below shows the relationship between standard free energy and the
equilibrium constant. Based on the previous picture and this table, we observe a
positive change in free energy (ΔG>0) if [C][D]/[A][B] at the moment is greater than
the equilibrium constant meaning that there are more products than reactants.
Therefore, this explains why the reverse reaction is favoured when ΔG>0.
4 Monday, January 11, 2016
Lecture 3. Energy in Biological Systems.
- Berg et. al. 430‐437.
- ATP is the currency of energy within the cell. It is called a high-energy phosphate
(high ∆G meaning that it has lots of free energy to release since the products are a
lot more stable than the starting material) compound for three main reasons:
• ADP and the inorganic phosphate have more resonance stabilization compared
to ATP which lowers the free energy.
• At pH 7, the triphosphate unit of ATP carries about four negative charges. These
charges repel one another because they are in close proximity. The repulsion
between them is reduced when ATP is hydrolyzed; therefore, electrostatic
repulsion lowers the free energy.
• More water can bind more effectively to ADP and P than can bind to the
phosphoanhydride part of ATP, stabilizing the ADP and P by hydration.
1 Monday, January 11, 2016
- For a reaction to occur, reacting species must pass through a higher energy state, the
“transition state”. Catalysts act to accelerate the rates of reactions by reducing the
ΔG between the reactants and the transition state. Enzymes are natural catalysts:
• Enzymes are speciﬁc catalysts for biological reactions (key&lock model). An
enzyme has a substrate binding site and a catalytic site; together they form the
• Enzymes act by binding to a reactant or reactants
(termed substrate[s]) in a way that reduces the
energy required to reach the transition state.
• Enzymes affect only the rates of the reaction –
other properties (e.g. K , ΔG) remain the same.
Enzymes return to their initial state once the
reaction is complete.
• E + S ES —> E + P
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- It is important to notice that he smaller the K m the higher the affinity the enzyme has
for the substrate.
- Photosynthetic organisms capture light energy and use it to:
• transfer the H atoms from water to acceptor molecules
• form molecular oxygen
• synthesize ATP from ADP and phosphate
• transfer H atoms from acceptor to carbons derived from CO to for2 glucose
• Net: 6 H 2 + 6 CO —>2C H O +66 12 6 2
- Other organisms feed on photosynthetic
organisms (directly, indirectly):
• Sugars, derived compounds formed through
photosynthesis acquired and oxidized to
water, carbon dioxide = respiration
• Respiration coupled to the formation of ATP
from ADP and phosphate (oxidative
• Ultimate electron acceptor is O ,2oxidation product is CO 2
- The ultimate source of all biological energy is sunlight.
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4 Monday, January 11, 2016
- In metabolism, there are lots of cycles:
• NADH and FADH2 reoxidized to NAD and FAD by the
• Electrons transferred to O to2form water (end of the
electron transport chain)
• Lots of free energy released: captured as proton
gradient across the mitochondrial inner membrane
• The free energy of the proton gradient used to drive the
synthesis of ATP from ADP and phosphate
• All this together is oxidative phosphorylation
- NAD /NADH and FAD/FADH high turnover rates: continually oxidized and reduced
during foodstuff oxidation. They are electron carriers.
• Metabolism is dynamic
• Foodstuffs constantly broken down to carbon dioxide and water
• NAD gets reduced to NADH then oxidized back to NAD with P + ADP —> ATP.i
ATP hydrolysis back to ADP and P used io drive cellular processes.
5 Monday, January 11, 2016
Lecture 4. Glycolysis.
- Berg et al., pp. 453‐469.
Glycolysis is the sequence of reactions that metabolizes one molecule of glucose to
two molecules of pyruvate with the concomitant net production of two molecules of
ATP. This is an anaerobic process taking place in the cytosol.
- Glycolysis is an example of fermentation (metabolic process with organic compound
as end product). In muscles, the product of fermentation is lactic acid, in yeast, it’s
ethanol. Glycolysis is an ancient metabolic pathway and is found in most organisms.
- In the ﬁrst stage of glycolysis, we invest 2 ATP but 4 ATP (2x2 ATP) is gained in the
second stage so there’s a net gain of 2 ATP.
- Kinases are enzymes that transfer γ phosphoryl group of ATP to acceptor.
6 Monday, January 11, 2016
7 Monday, January 11, 2016
1. Hexokinase uses an ATP to phosphorylate glucose since glucose 6-phosphate
cannot pass through the membrane because it is not a substrate for the glucose
transporters, and the addition of the phosphoryl group acts to destabilize glucose,
thus facilitating its further metabolism.
2. The isomerization of glucose 6-phosphate to fructose 6-phosphate is a conversion
of an aldose into a ketose. The reaction catalyzed by phosphoglucose isomerase
takes several steps because both glucose 6-phosphate and fructose 6-phosphate
are present primarily in the cyclic forms. The enzyme must ﬁrst open the six-
membered ring of glucose 6-phosphate, catalyze the isomerization, and then
promote the formation of the ﬁve-membered ring of fructose 6-phosphate.
3. Fructose 6-phosphate is phosphorylated at the expense of ATP to fructose 1,6-
bisphosphate. This reaction is catalyzed by phosphofructokinase (PFK) and is
very important since it is a rate regulating step.
4. The newly formed fructose 1,6-bisphosphate is cleaved into glyceraldehyde 3-
phosphate (GAP) and dihydroxyacetone phosphate (DHAP) by the enzyme
aldolase. Glyceraldehyde 3-phosphate is on the direct pathway of glycolysis,
whereas dihydroxyacetone phosphate is not. These compounds are isomers that
can be readily interconverted: dihydroxyacetone phosphate is a ketose, whereas
glyceraldehyde 3-phosphate is an aldose.
5. The isomerization of these three-carbon phosphorylated sugars is catalyzed by
triose phosphate isomerase. At equilibrium, 96% of the triose phosphate is
dihydroxyacetone phosphate. However, the reaction proceeds readily from
dihydroxyacetone phosphate to glyceraldehyde 3-phosphate because the
subsequent reactions of glycolysis remove this product.
The preceding steps in glycolysis have transformed one molecule of glucose into
two molecules of glyceraldehyde 3-phosphate, but no energy has yet been
extracted. On the contrary, thus far, two molecules of ATP have been invested. We
come now to the second stage of glycolysis, a series of steps that harvest some of
the energy contained in glyceraldehyde 3-phosphate as ATP. The initial reaction in
this sequence is the conversion of glyceraldehyde 3-phosphate into 1,3-
bisphosphoglycerate (1,3-BPG), a reaction catalyzed by glyceraldehyde 3-
phosphate dehydrogenase. This reaction can be viewed as the sum of two
processes: the oxidation of the aldehyde to a carboxylic acid by NAD andthe
joining of the carboxylic acid and orthophosphate to form the acyl-phosphate
8 Monday, January 11, 2016
7. 1,3-Bisphosphoglycerate is an energy-rich molecule with a greater phosphoryl-
transfer potential than that of ATP. Thus, 1,3-BPG can be used to power the
synthesis of ATP from ADP. Phosphoglycerate kinase catalyzes the transfer of the
phosphoryl group from the acyl phosphate of 1,3-bisphosphoglycerate to ADP. ATP
and 3-phosphoglycerate are the products. The formation of ATP in this manner is
referred to as substrate-level phosphorylation because the phosphate donor, 1,3-
BPG, is a substrate with high phosphoryl-transfer potential. We will contrast this
manner of ATP formation with the formation of ATP from ionic gradients.
8. The position of the phosphoryl group shifts in the conversion of 3-phosphoglycerate
into 2-phosphoglycerate, a reaction catalyzed by phosphoglycerate mutase. In
general, a mutase is an enzyme that catalyzes the intramolecular shift of a chemical
group, such as a phosphoryl group.
9. The dehydration of 2-phosphoglycerate introduces a double bond, creating an enol.
Enolase catalyzes this formation of the enol phosphate phosphoenolpyruvate
(PEP). This dehydration markedly elevates the transfer potential of the phosphoryl
group. An enol phosphate has a high phosphoryl-transfer potential, whereas the
phosphate ester of an ordinary alcohol, such as 2-phosphoglycerate, has a low one.
The ∆G of the hydrolysis of a phosphate ester of an ordinary alcohol is -13 kJ/mol,
whereas that of phosphoenolpyruvate is -62 kJ/mol.
10. The phosphoryl group traps the molecule in its unstable enol form. When the
phosphoryl group has been donated to ATP, the enol undergoes a conversion into
the more stable ketone —> pyruvate. The virtually irreversible transfer of a
phosphoryl group from phosphoenolpyruvate to ADP is catalyzed by pyruvate
9 Monday, January 11, 2016
-The conversion of glucose into two molecules of pyruvate has resulted
in the net synthesis of ATP. However, an energy-converting pathway
that stops at pyruvate will not proceed for long, because redox balance
has not been maintained. NAD must be regenerated for glycolysis to
proceed. Thus, the ﬁnal process in the pathway is the regeneration of
NAD through the metabolism of pyruvate.
- When the lactic acid builds up, the pH in the cell decreases and muscles get tired.
10 Monday, January 11, 2016
Lecture 5. Regulation of glycolysis and gluconeogenesis.
- Berg et al., pp. 472‐88.
- During glycolysis, enzymes that catalyze irreversible steps are allosterically regulated:
• Phosphofructokinase ( PFK) is inhibited by ATP, citrate, H , and activated by
AMP , fructose-2,6-bisphosphate (F-2,6-BP).
• Hexokinase is inhibited by its end product —> glucose-6-phosphate.
• Pyruvate kinase is is inhibited by ATP and activated by fructose-1,6-bisphosphate.
- PFK is the key regulator of this metabolic pathway. This
enzyme is allosterically regulated and it has 4 identical
subunits and each subunit has a catalytic site which can
bind ATP and fructose-6-phosphate.
• PFK has 2 ATP binding sites: 1 catalytic, 1 regulatory.
The graph on the right shows the enzyme’s activity
depending on the concentrations of ATP and
• ATP bound at the regulatory site lowers afﬁnity for
fructose-6-phosphate. AMP reverse inhibition by ATP;
AMP acts as a positive regulator. There’s an enzyme
called adenylate kinase (picture on the right) and it
creates AMP; when AMP concentration is high, the
cells know that there is a huge lack of ATP and will
turn phosphofructokinase back on.
• Citrate (citric acid) enhances ATP inhibition (liver only). TCA occurs in the
mitochondria, if it ends up in the cytosol, it means that there is too much ATP.
• PFK is also inhibited by low pH (high proton concentration) and prevents excessive
accumulation of lactic acid.
• Fructose-2,6,-bisphosphate is a key regulator of PFK (liver only). When glucose
levels are high in the blood, fructose-6-phosphate levels are also high, which
stimulates a second enzyme called phosphofructokinase-2 (PFK2). PFK2 creates
fructose-2,6,-bisphosphate and this molecule decreases the inhibitory effect of ATP,
in other words, it increases PFK’s afﬁnity for fructose-6-phosphate and enhances
11 Monday, January 11, 2016
- Additional regulation: hexokinase and pyruvate kinase:
• At rest, glucose-6-phosphate will accumulate and shut off hexokinase. Glucose-6-
phosphate can also be converted into glycogen for storage. PFK is also shut off by
high levels of ATP which also shuts off pyruvate kinase.
• During exercise, there is less ATP present and more AMP. Hexokinase will be
active since AMP activates PFK which stimulates hexokinase to produce more
glucose-6-phosphates. The high level of fructose-1,6-bisphophate also stimulates
the activation of pyruvate kinase.
- Once we get to pyruvate, it all depends on how much oxygen is present. If there is
enough oxygen (e.g. jogging), pyruvate will go through TCA, if there is low oxygen
levels (e.g. sprint), fermentation and formation of lactate will take place.
12 Monday, January 18, 2016
Lecture 6. Glycogen metabolism .
- Berg et al., pp. 489‐492; 615‐635; Lodish et al., pp. 699‐703.
- The “ Warburg” effect is a phenomenon
concerning biological oxidation. Rapidly growing
tumours metabolize glucose to lactate even
when ample oxygen is present because
tumours grow faster than blood vessels that
feed them: environment becomes oxygen poor
– hypoxia. Hypoxia activates HIF transcription
factor. Active HIF switches on genes encoding
glycolytic enzymes. Active HIF increase
expression of VEGF – stimulates blood vessel
- Gluconeogenesis is somewhat the opposite of glycolysis; non-carbohydrate
precursors (lactate, some amino acids, glycerol) are converted into glucose. This
pathway takes place mainly in the liver and is important during extended exercise
and fasting in order to keep blood glucose levels up. The brain is dependent on
glucose, it doesn’t take any other form of energy!
- Gluconeogeneis is not simply the reverse of glycolysis, not all the steps in the
pathway are reversible. The picture below boxed the irreversible steps.
1 Monday, January 18, 2016
- In fact, in the gluconeogenesis pathway, the three irreversible steps of glycolysis are
replaced by four other steps.
• 1. Pyruvate —> Oxaloacetate (OAA)
• 2. OAA —> PEP
• 3. F-1,6-BP —> F-6P
• 4. 6-6P —> glucose
• Some amino acids and lactate —> pyruvate
• Other amino acids —> OAA
• Glycerol (from lipids) —> DHAP
- Pyruvate carboxylase adds a carboxyl group to
pyruvate and converts Pyruvate to OAA. This
enzyme is located in the mitochondria, it contains
biotin necessary for carboxylation and it required
acetyl CoA although it is not a substate.
- PEP carboxykinase: OAA+GTP—>PEP+GDP+CO2.
PEP carboxykinase is cytosolic (contained in the
cytosol) In order to convert pyruvate to PEP, there is
ﬁrst a carboxylation and followed by a decarboxylation.
2 Monday, January 18, 2016
- Fructose 1,6 Bisphosphatase converts F-1,6-BP —> F-6P + P. It is inhibiied by
AMP and F-2,6-BP but activated by citrate. It is important that fructose 1,6
bisphosphatase and PFK aren’t operating simultaneously since it will burn ATP for
absolutely no reason.
- Fructose-6-phosphate is easily converted into glucose-6-phosphate (reversible step).
Glucose-6-phosphatase then converts G-6P —> G + P. Glucose-6-ihosphatase is
only found in tissues (liver, kidney) that regulate blood glucose levels. It is important
to regulate free glucose levels. Remember that glucose can pass through the cell
membrane but not the phosphorylated form.
- Conditions that favour glycolysis inhibit gluconeogenesis
and vice versa. Glycolysis is catabolic (breakdown) and
energy yielding while gluconeogensis is anabolic (build-
up) and energy consuming.
- During intensive exercise, pyruvate
made faster in muscle than citric acid
cycle can oxidize it —> lactate
production. Lactate in blood is then
taken up by liver. The lactate converted
back into glucose in liver through
gluconeogenesis. The picture above is
a simple illustration and the picture on
the right is more complete. Important to
know that the cardiac muscle is very
rich in mitochondria.
3 Monday, January 18, 2016
- Glucose is stored as glycogen. The precursor of glycogen is uridine-diphosphate
glucose. Breaking glycogen down, requires an inorganic phosphate group.
4 Monday, January 18, 2016
- Blood glucose must be tightly regulated. Both glucagon and insulin are hormones
that regulate it.
• Insulin released from the
pancreas when blood glucose is
high, stimulates synthesis of
glycogen from glucose.
• Glucagon released from pancreas
when blood glucose is low,
stimulates the breakdown of
glycogen to glucose.
• M u s c u l a r a c t i v i t y o r i t s
anticipation stimulates release of
epinephrine (adrenalin) from
- Glucagon binding to receptor on liver cell, or epinephrine binding on muscle cell
activates adenylate cyclase: ATP —> cAMP + PP. i
5 Monday, January 18, 2016
- READ BOOK!
- Elevated cAMP levels activate protein kinase A (PKA).
6 Monday, January 18, 2016
Lecture 7. The tricarboxylic acid (TCA) cycle .
- Berg et al., pp. 497‐516.
- In order to regenerate NAD when there is no
oxygen, our body undergoes lactic acid
fermentation. But pyruvate can be oxidized if
oxygen is present.
- Pyruvate has to be ﬁrst converted into carbon
dioxide and acetyl CoA to enter the tricarboxylic
• Carbohydrates —> pyruvate —> acetyl CoA
• This pathway allows recovery of much greater portion
of free energy than glycolysis and occurs in the
• The mitochondria has two membranes (outer and
inner), there is an inter membrane space in between
them. The inner membrane encloses the mitochondrial
matrix where the Kreb’s cycle takes place.
- Acetyl CoA has a sulfhydryl group that binds (thioester bond) to the acetyl groups
and that bond stores a lot of energy.
The acetyl group of acetyl CoA completely oxidizes to two CO .
• The 8 electrons from acetyl are transferred to NAD+ and FAD.
NADH and FADH forme2 during cycle
reoxidized to NAD+ and FAD through action
of respiratory or electron transport chain.
- A proton gradient is created within the
intermembrane of the mitochondria with the
electrons from NADH and FADH and 2
transferring the low energy electrons to
oxygen releasing water. Then, the gradient
could be discharged through oxidative phosphorylation when protons ﬂow back and
pass by ATP synthase.
7 Monday, January 18, 2016
- The ﬁrst step after glycolysis, if oxygen is present, is to oxidize pyruvate in the
mitochondria. It is an irreversible reaction and is catalyzed by pyruvate
dehydrogenase complex. The complex has over three distinct enzymes associated in
a huge protein/coenzyme complex (60 polypeptide chains)
The three distinct coenzymes derived from vitamins and they are E1 (thiamine
pyrophosphate), E2 (lipoic acid) and E3 (FAD).
• Pyruvate + CoA + NAD+ —> Acetyl CoA + CO + NADH + H 2 +
- Because the pyruvate dehydrogenase reaction is irreversible, it is highly regulated.
• It is allosterically inhibited by end products (NADH and acetyl CoA).
• It is switched off by phosphorylation; increased NADH/NAD+ ratios, acetyl CoA/
CoA ratios or ATP/ADP ratios promotes phosphorylation.
• An accumulation of substrates (pyruvate and ADP) promotes dephosphorylation.
- In the next step, TCA cycle begins (overall picture in following pages).
1. Acetyl CoA binds to oxaloacetate (4 carbon compound) forming citrate (citric
acid) which is a tricarboxylic acid (3 carboxyl groups) with the enzyme citrate
synthase. It is the hydrolysis of the thirster bond that releases the energy used
to drive the reaction. Therefore, the equilibrium lyes far to the citrate formation.
2. The enzyme aconitase removes a water molecule and re-adds it forming an
isomer called isocitrate; the hydroxyl has been moved.
8 Monday, January 18, 2016
3. Isocitrate dehydrogenase then oxidizes isocitrate’s hydroxyl group into a
carbonyl. A NAD+ is also changed into NADH. Then, a carboxyl group is
released forming alpha-ketoglutarate. This type of process is called an oxidative
In the next step, we go from alpha-ketoglutare (5 carbon dicarboxylic acid) to
succinate (4 carbon dicarboxylic acid).
alpha-ketoglutarate + GDP + Pi —> succinate + GTP.
1. Alpha-ketoglutarate dehydrogenase removes a terminal carboxyl and
forms a thioester with CoA. A NADH is produced. This reaction has the same
mechanism as pyruvate dehydrogenase. This step is inhibited by NADH and
2. Succinyl CoA synthetase hydrolyses the thioester bond to release CoA.
There are different form of this enzyme depending where it is. At the same
time, the energy is captured to phosphorylate GDP —> GTP or ADP —> ATP.
depending where the enzyme is located once again.
9 Monday, January 18, 2016
5. Since it is a cycle, succinate has to be reconverted into oxaloacetate.
1. Succinate dehydrogenase located in the inner membrane converts it into
fumarate and electrons go to form a FADH . 2
2. Fumerase then adds a water across the double bond converting fumarate to
3. Malate dehydrogenase completes the cycle but equilibrium favours malate.
therefore it is not a very efﬁcient reaction.
10 Monday, January 18, 2016
- The ﬁrst reaction of the cycle lyes far to citrate (very negative free energy change)
whereas the last reaction of the cycle lyes far to the malate (very positive free energy
change). Therefore, the concentration of oxaloacetate remains low within the
mitochondria matrix. In certain cases, oxaloacetate’s concentration can be so low
that it’ll limit the rate of TCA.
- Both decarboxylation steps (3 &4) have negative free energy change, hence the
overall standard free energy change is low for the cycle.
- Succinate dehydrogenase, unlike the other proteins, this enzyme is part of a
respiratory complex of the inner mitochondrial membrane, it does not rely in the
matrix but in the membrane.
AcetylCoA+3NAD +FAD+GDP+P → CoA+2CO +i ADH+FADH +GTP 2 2
11 Monday, January 18, 2016
Lecture 8. Regulation of the TCA.
- Berg et al., pp. 516‐521; 639‐648.
Isocitrate dehydrogenase’s activity is important, high change in free energy. It is
reduced by ATP and NADH and activated by ADP and NAD+.
- Alpha-ketoglutarate dehydrogenase complex is also inhibited by ATP and NADH and
- The availability of oxaloacetate is the limiting factor of this cycle.
- The intermediate molecules part of the cycle are very important and many are
precursors of formation of other biomolecules. For example, citrate can be exported
out of the mitochondria into the cytoplasm and used to synthesize fatty acids.
12 Monday, January 18, 2016
- Fatty acid oxidation can occur in mitochondria or peroxisomes.
• Fatty acids —> Fatty Acyl CoA, it gets “activated”. This step uses ATP and occurs
within the cytoplasm.
• Pathways are similar in both compartments (mitochondria and peroxisome).
• Products are Fatty Acyl CoA - 2 carbon + Acetyl CoA.
13 Monday, January 18, 2016
• In mitochondria, FADH 2 and NADH are oxidized by
respiratory chain: Acetyl CoA enters TCA where 2 CO 2
• In peroxisomes, FADH red2ces O toH O a2d acet2l2
CoA gets exported.
- Ketone bodies are produced by the liver from fatty acids
during periods of low food intake. In other words, they exist
when gluconeogeneis and fatty oxidation occur
simultaneously, insufﬁcient oxaloacetate to react with
acetyl CoA produced.
- These ketone bodies are converted into acetyl-CoA which
then enters the citric acid cycle and is oxidized in the
mitochondria for energy.
14 Monday, January 25, 2016
Lecture 9. Metabolism and physiology .
- Lodish et al., 473‐503.
- When gluconeogeneis (catabolic pathway) and fatty acid oxidation (anabolic pathway)
occur simultaneously, there isn’t enough oxaloacetate to react with acetyl CoA.
Therefore, ketone bodies are produced.
- A consequence is the fact that ketone bodies are relatively strong acids and will lower
the pH in the blood. Dangerous is pH is too low in the brain.
• Oxaloacetate is an intermediate in gluconeogenesis.
• When acetyl CoA concentration is too high in the mitochondrial matrix, it can react
with itself and can form betahydroxybutyrate and acetoacetate.
• This occurs in the liver and it will release these compounds in the blood stream.
- This situation happens when one
• The body prefers
carbohydrates as a source of
energy. If you don’t eat all day,
the stored glycogen is used up
but the body needs to keep
glucose in the blood for the
brain to function.
• In absence of dietary
limited and fats are going in
the liver to extract energy.
• Since glucose is needed, liver
oxaloacetates are directed to
the formation of glucose.
• Some dietary amino acids
converted into glucose by liver
(gluconeogenesis) while other dietary amino acids are converted to acetyl CoA.
1 Monday, January 25, 2016
• Hence, there is an increase in the reliance of fats as energy source — breakdown
of adipose tissue. Therefore, fatty acid oxidation and gluconeogenesis are
occurring at same time .
• Ketone bodies are produced by liver and exported in the bloodstream
• In extreme conditions (starvation) brain adapts by allowing ketone bodies across
blood brain barrier.
- Ketone bodies are also present when one has diabetes.
• There is a lot of glucose in the blood but cells aren’t able to take it all up because
no insulin is present. Thus, no glycolysis going on in the tissues; there is high blood
glucose but it cannot get into the cells.
• The liver oxaloacetate is hence directed to the formation of glucose.
• Some dietary amino acids converted into glucose by liver (gluconeogenesis) while
other dietary amino acids are converted to acetyl CoA.
• Therefore lipids are broken down and released into the bloodstream. All the fatty
acids are broken down into acetyl CoA and ketone bodies.
• The brain once again adapts by allowing ketone bodies to cross the blood brain
- Both carbohydrates and fats can be used provide energy but carbohydrates are
• Glucose is only source of energy for the brain since there is a blood/brain barrier.
• Red blood cells have no mitochondria so have to rely on glycolysis.
• High intensity exercise (sprint) driven almost exclusively by anaerobic glycolysis.
The maximum human sprint distance (full speed) is about 200 meters and therefore
pacing is critical for longer distance races.
- Scenario of 800m race. He starts fast, he slows down, he feels good, he’s hanging on
towards the end and can barely walk after the race. What happened?
• Lactic acid build up early. Slow down in pace allowed for more aerobic metabolism
• Last 100m: need for muscle ATP acute but lactic acid build up leads to increase in
muscle proton concentration which inhibits PFK. Glycolysis stops and muscles stop
2 Monday, January 25, 2016
- Scenario of marathon: about 75% of calories come from carbohydrate reserves and
25% from fats.
• Maximum carbohydrate energy storage is around 2000 calories (good for 20 miles)
• As the race goes on, carbohydrates becomes depleted. If carbohydrate gets
depleted before race ﬁnishes you hit the wall.
• Fatty acids cannot supply the energy without carbohydrates since oxaloacetate
limits the tricarboxylic acid cycle. The body has to keep doing gluconeogenesis to
replenish oxalocatate but the brain and red blood cells need glucose. Hence, there
is no way to provide muscles with adequate ATP to maintain pace.
- The Atkins diet is basically cutting off carbohydrates forcing the body to use stored fat
as energy source.
• The proteins provide a source of oxaloacetate and some glucose through
• A downside of this is bad breath because ketone bodies are produced.
• It is a state of constant ketosis which is not good longterm since ketone bodies do
acidify the blood.
3 Monday, January 25, 2016
- Phospholipids consist of a glycerol which has 3 hydroxyl groups. Two of those groups
are attached to two fatty acid chains via ester linkage and a third terminal one is
attached to a phosphate (phosphatidic acid, PA).
- Phosphates are usually linked to another group. For example, choline in phosphatidyl
- Glycerol phosphate portion constitutes polar head, water loving.
- Phospholipids are the major lipid component of cell membranes
- The amphipathic phospholipids instantly form a phospholipid bilayer. These bilayers
are permeable to small non-polar molecules e.g. CO2 and O2. They are partly
permeable to water and impermeable to charged molecules, ions and larger
uncharged molecules such as glucose.
- Cellular membranes consist of proteins embedded in a phospholipid bilayer.
- Most substances require special transport protein to pass across membranes.
4 Monday, January 25, 2016
Lecture 10. Metabolic integration.
- Berg et al., pp. 654‐66; 791‐810.
Movement left to right is energetically favourable; it is
entropy driven. The opposite from right to left is
• To move a molecule against gradient (uphill) we must expend energy: G > 0
• Movement down a concentration (downhill) gradient releases energy: G < 0
- Passive diffusion is a downhill movement of a substance across a lipid bilayer.
• Smaller non-polar or mildly polar molecules can diffuse across cell membranes.
• The rate of passage across membrane is determined by concentration gradient,
permeability coefﬁcient; the more hydrophobic, the higher permeability coefﬁcient.
• For example: benzene (non polar) will diffuse faster than ethanol which is faster
than ammonia or alanine (charged on both sides of the amino acid body even at
- There are different types of membrane transport
proteins: most substances require membrane
transport protein to enter or leave the cell.
• ATPase pumps — active transport uphill coupled
to ATP hydrolysis.
• Ion channels and uniporters — transport downhill
such as facilitated diffusion.
• Symporters and antiporters — uphill movement
of one molecule coupled to downhill movement of
- GLUT1 is an important protein (for red blood cells
speciﬁcally). It has a glucose binding site, the binding
leads to a conformational change ﬂipping the channel towards the interior of the cell.
Then the glucose is phosphorylated and gets trapped.
5 Monday, January 25, 2016
- Transporters have something similar to a K m
GLUT1 (see picture on the right). The normal
concentration of glucose in the blood is around
5 mM; hence, GLUT1 is nearly operating at
nearly 75% of its maximum capacity. GLUT3 is
very similar to GLUT1, it is found in neuronal
- GLUT2 is present in the liver but is different
than GLUT1 and GLUT3. GLUT2 in the liver
only starts to take up glucose when the
concentrations are very high after a meal for example. This enzyme has a relatively
high K malue and it can also work in reverse and pump glucose out of the liver when
blood glucose levels are low.
- The movement is ions across membranes is more challenging than an uncharged
molecule; it has to move against both a charge and a concentration gradient which
might even be opposed at time. Z is the charge, F is the Faraday constant and V is
- In the opposite direction, log C 2C 1ill give a
number smaller than zero and yielding an
negative free energy.
- Cells in general maintain a potential gradient
that is negative on the inside. Potassium
concentrations are relatively high within the
cell and sodium concentrations are relatively
6 Monday, January 25, 2016
- Three ion speciﬁc channels are illustrated on the
right. a) doesn’t allow anything to pass through, b)
only allows the ﬂow of Na ion and c) only allow
potassium ions. Therefore, ions move down the
concentration gradient and stops when the energy
term from charge change (ZFV) is equal to the
term from concentration change (2.3 RT log C /C ).
- The picture above illustrates that the entry of
sodium into the cell is energetically favourable.
7 Monday, January 25, 2016
- There are many ATP-powered ion pumps divided in a few classes (P-class pumps, V-
class proton pumps, F-class proton pumps & ABC superfamily).
- P-class pumps transports only ions and there are 2 alpha (usually) and 2 beta
subunits, it can exist as tetramers. It is the alpha subunit is involved in transport and
• An example is the animal Na /
K ATPase (sodium potassium
pump). It needs to keep
potassium concentrations high
within the cell to synthesize
proteins and sodium
concentrations low. For each
ATP hydrolyzed, it pumps 3 Na
+out and 2 K in. It is also
inhibited by oubain and
8 Monday, January 25, 2016
- V-class H pumps are similar in structure to
the F-class proton pumps which are involved in
oxidative phosphorylation and photosynthesis.
• They pump protons into lysosomes and
vacuoles. Enzyme is not phosphorylated
during reaction cycle.
• In order to compensate for the charge
differential, these pumps are coupled to a
chloride ion channel for effective acidiﬁcation
of the lumen.
- ABC transporters (ATP Binding Cassette)
have two transmembrane and two ATP binding
• These proteins are very common in bacteria.
For example, bacteria permeases allow entry
of key nutrients such as some amino acids.
• An example is mammalian MDR (multiple
drug resistance) transporters: it transports
drugs to the exterior of the cell and the
membrane orientation of the protein changes.
• Another example is the CFTR protein: cystic
ﬁbrosis transmembrane regulator is a
chloride channel protein in lungs and other
9 Monday, January 25, 2016
Lecture 11. Biological redox reactions (oxidative phosphorylation) .
Berg et al., pp. 525‐531.
- NADH and FADH are f2rmed during cycle re-
oxidized to NAD and FAD through action of
respiratory or electron transport chain.
-Respiratory chain transfers electrons
to O 2o form H O.2Coupled to
formation of H gradient across inner
- The discharge of gradient is coupled to ATP synthesis (oxidative phosphorylation)
- NADH and Succinate come in and electrons
are transferred to lots of electron carriers.
- Redox reactions require an electron donor and
electron acceptor (NAD and FAD).
-In the electron transfer chain, each
transfer from carrier to carrier is a redox
Succinate + FAD —> fumarate + FADH . 2
- It can be considered as two half reactions.
Succinate —> fumarate + 2e- + 2H (oxidation)
• FAD + 2e- + 2H —>+FADH (reduc2ion)
10 Monday, January 25, 2016
The next reaction in the chain involves a non-heme ion (NHI).
FADH + 2Fe 3+ (NHI) —> FAD + 2Fe 2+ (NHI) + 2H +
- Therefore, oxidation is a gain of H/H character or a loss of elect