BIO 361

Exam 4 Lecture Notes

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Lecture 29: Electron Transport I

(pgs. 2-3)

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Lecture 30: Electron Transport II

(pgs. 4-6)

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Lecture 31: Electron Transport III

(pgs. 7-10)

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Lecture 32: Oxidative Phosphorylation

(pgs. 11-13)

MK

Lecture 33: Fatty Acid Degradation I

(pgs. 14-18)

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Lecture 34: Fatty Acid Degradation I

(pgs. 19-22)

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Lecture 35: Fatty Acid Biosynthesis

(pgs. 23-26)

MK

Lecture 36: Cholesterol Metabolism

(pgs. 27-31)

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Lecture 37: Metabolic Regulation I

(pgs. 32-36)

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Lecture 38: Metabolic Regulation II

(pgs. 37- 39)

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Lecture 39: Metabolic Regulation III

(pgs. 40 -42)

Daniel S. McCaffrey
Abbreviations: ETC: Electron Transport Chain; IMS: Intermembrane Space; IMM: Inner
Mitochondrial Membrane; OMM: Outer Mitochondrial Membrane; OAA: Oxaloacetate;
PKA: Protein Kinase A; PK: Phosphorylase Kinase; AC: Adenylate Cyclase

BIO 361 Exam 4 Review

Daniel S. McCaffrey

Lecture 29 (Friday, November 11th, 2016): Electron Transport I

12 electron pairs are passed to NAD and FAD in the mitochondrion from glucose oxidation in the cytosol
(glycolysis). These electrons are passed between 4 mitochondrial redox centers, each an enzyme complex,
before being used to reduced oxygen to water.
During electron transport, PROTONS are expelled from the mitochondrion into the cytosol, establishing
an electrochemical proton gradient. This gradient is what drives ATP production by ATP synthase in the
energy that it stores.
The mitochondrial intermembrane space is ultimately continuous with the cytosol: they are equivalent in
ion concentration. The inner mitochondrial membrane is the true division between the mitochondrial
matrix and the cytosol.
The inner mitochondrial membrane is loaded with protein: some completely transverse the membrane,
others do not. The IMM is where the action lies.
The controlled impermeability of the IMM is highly regulated and allows for ion gradients to be
established across it, facilitating the compartmentalization of reaction pathways as well.
Electron Transport Chain (ETC) oxidizes reduced NADH and FADH2 progressively. Each enzyme
complex is associated with a specific reduction potential.
The ultimate endgame of the ETC is the reproduction of oxidized NAD+ and FAD and H2O by the
reduction of O2. The deltaG values across the reaction schemes are generally favorable, but ATP cannot
be generated from them in a concerted reoxidation pathway. Reoxidation is therefore done in a
STEPWISE fashion. This succeeds in:

Maximizing efficiency in the pathway

Controlling/minimizing the success of side-pathways which can produce free-oxygen radical
species and are therefore dangerous to cell health. They are also culprits in reducing the overall
efficiency of the ETC pathway: energy which would normally fuel these side reactions is instead
trapped by the enzyme complexes and later repurposed for ATP synthesis.

ETC reactions do NOT directly cause substrate-level phosphorylation (ATP synthesis). They are redox
reactions which convert/store energy into/in a PROTON GRADIENT between the matrix and the
intermembrane space/cytosol; this gradient drives ATP synthesis by ATP synthase.
Each ETC complex contains multiple proteins; all but Complex II completely span the IMM, with
elements in both the matrix and the IMS.

Complex II features succinate dehydrogenase (from the Krebs Cycle). It has a good reach into the
matrix but a poor one into the IMS.

ALL of the ETC complexes have elements which are responsible for proton and electron transport across
the IMM EXCEPT for Complex II, which only assists in electron transport and provides supplemental
energy.
2

COMPLEX I: NAH oxidase, coQ reductase (with proton movement).

COMPLEX II: FADH2 oxidase, coQ reductase.
COMPLEX III: coQ oxidase, cytochrome C reductase (with proton movement).
COMPLEX IV: cytochrome C oxidase, O2 reductase (to H2O) (with proton movement).
The redox reactions themselves are NOT associated directly with proton movements: certain proteins
connect these reactions to the successful movement of protons.
ETC redox reactions also generate (as aforementioned) reoxidized NADH and FADH2.
ETC redox reactions occur WITHIN the complexes at other centers to avoid inefficiency. These other
centers are (generally) Fes (iron-sulfur) complexes/polymers.
FeS complexes capture the energy of the reduction potentials of the complexes.
coQ is a very HYDROPHOBIC and is intimately confined to the lipid bilayer of the IMM. Complex II
oxidizes the FADH2 produced by succinate dehydrogenase in order to later reduce coQ.

Complex I is large; it utilizes FMN (Flavin-mononucleotide) instead of FAD. 6-7 FeS clusters act as
supplemental redox centers for electron transport, too.
NADH can ONLY participate in 2-electron transfer reactions. FMN and coQ split-up the 2 electrons
from a single NADH molecule across two sequential 1-electron reactions:

coQ 1 electron semiquinone radical form 1 electron ubiquinol (2 electron form)

FMN 1 electron

FMNH radical form

1 electron FMH2 (2 electron form)

Both radical forms are (relatively) STABLE. They allow for single-electron transfer to the 1-electron
acceptors of Complex III (which conducts 1-electron reduction of cytochrome irons).
The linear arrangement of the FeS complexes facilitate electron passage from one end of the EMM to the
other.
Rotenone and Amytal are famous selective INHIBITORS of Complex I: they shut down the reoxidation
of NADH.

Complex II passes electrons from succinate to coQ. It utilizes an FAD group and 3 FeS clusters. The FeS
complexes do NOT come close to the edges of the IMM.
COMPLEXES I AND II REPRESENT ALTERNATIVE MECHANISMS FOR coQ REDUCTION:
THEY DO NOT OPERATE IN SERIES. coQ acts as an electron collection point.

Lecture 30 (Monday, November 14th, 2016): Electron Transport II

Recap: Transfer of electrons from NADH and FADH2 to O2 (generating H2O) occurs in a STEP-WISE
fashion across four (4) complexes in the IMM, capturing an energetic potential by means of a
proton gradient across the IMM, established with proton movement across the IMM.
High energy intermediates do NOT store energy potentials in this pathway.
Complexes I and II BOTH possess internal flavins (FMN/FAD); Complex II utilizes a
cytochrome b group separate from its redox pathway.
Complex I uses NADH; Complex II uses FADH2: they are alternative routes and do not operate
in series.
Complex II does NOT span the entire IMM: it has a better reach into the matrix than the IMS but
does not facilitate proton movement at all.
The flavins (FMN and FAD) have the capacity to exist in 3 different oxidation states (in order of
progressive reduction): quinone semiquinone hydroquinone
coQ (the substrate for both complexes I and II) can also exist in three forms analogous to these.
Complex Is last FeS cluster delivers electrons to coQ; Complex II also utilizes a linear chain of
FeS clusters/proteins to facilitate electron passage.
How about electron movement? (Only by Complex I) It doesnt take place by means of
protonation/deprotonation of any of the centers involved in the complexs redox pathway.
RATHER, the protons moved by Complex I move as a result of CONFORMATIONAL
CHANGES in some amino acid components of other proteins of the complex. Protons are
picked up from the matrix and deposited into the IMS when conformational changes in the
proteins induce localized pK modifications which affect the side chains capacity for
proton-retention.
These pK changes arent associated directly with redox reactions: Redox Reactions
Conformation Changes Proton Pick-Up/Put-Down (transfer).
Recall that coQ is completely hydrophobic and confined to float freely between complexes I and
II inside the lipid bilayer of the IMM.

Complex III:

two coQ binding sites

two Heme+ Cytochrome b groups: bH heme (HIGH potential) and bL heme (LOW
potential)
several FeS complexes: two important ones (the complex is a homodimer) can utilize
histidines (the ISP or Rieske Center); they also serve to deliver electrons to the
cytochrome c1s.
two Heme/Cytochrome C1 groups which donate electrons to a water soluble protein in
the IMS: CYTOCHROME C, which can closely associate with cytochrome c1 in
Complex III BUT can also diffuse into the IMS to reach Complex IV.
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Cytochrome C is the second electron carrier in the ETC pathway (coQ is the first)
As coQ diffuses from complexes I and II, it is fully reduced. Complex III fully oxidizes it, and the
two electrons associated with each H of the fully reduced coQH2 form are split-up into two different
pathways.
Stigmatellin INHIBITS Complex III at the Q0 binding site close to the IMS
Antimycin A INHIBITS Complex III at the Qi binding site close to the matrix.
The Q-CYCLE allows coQH2 to reduce two molecules of cytochrome C, a 1-electron carrier.

Cycle 1: fully reduced

coQH2 loses two protons to
the IMS as it binds to the Q0
binding site. 1e- is
transferred to the ISP (FeS
Protein) or Rieske Center,
which shuttles it to
cytochrome c1, which
reduces the first cytochrome
C in the IMS. The other eremains a part of the
semiquinone form and is
soon separated from it and
then shuttled across the
Heme/cytochrome b
complexes to the Qi binding
site, where coQ is reduced to
back to the semiquinone
form.
Cycle 2: all of the action
which takes place at the Q0
binding site in Cycle is
repeated a second time: two
more protons are sent into the
IMS and another cytochrome
C is reduced. This time at the
Qi binding site, the
semiquinone form produced
in Cycle 1 is reduced to
coQH2 with the e- shuttled
across the Heme/cytochrome
bs and 2 protons taken from
the MATRIX.
5

Complex III/the Q-Cycle reduces cytochrome C (coQ ISP cytochrome c1) and moves protons,
using coQ as an H+ carrier, from the matrix to the IMS.
TWO turns of the cycle are required in order to re-reduced the semiquinone coQ form back to coQH2.
A 2H+ Gradient is propagated by the two turns of the Q-Cycle: 4H+ are sent to the IMS; 2 H+ are
imported from the matrix.

Complex IV:

reduced by the mobile electron carrier cytochrome C.

CN- (cyanide) is a potent inhibitor.
two Heme/cytochromes: a and a3
two Cu 2+ centers.
Overall reaction REOXIDIZES cytochrome C and REDUCES O2 to two molecules of
H2O.

BIG, multi-subunit complex, fully spanning the IMM. This allows it to donate protons across the IMM in
the same way that Complex I does: amino acid side chains with conformational-change-altered pKs
allow for H+ to be deposited across the IMM.
Complex IV accepts electrons from cytochrome C; these are then passed from CuA complex and hemes a
and a3 to the CuB, where the heme a3 iron splits an O2 molecule into two atoms of oxygen (this is the O2
reducing center). Two OH (hydroxide) anions are produced; they pick up protons to become H2Os.
Cytochrome C CuA complex Heme a Heme a3 + CuB +O2 (2)-OH 2H2O

Lecture 31 (Wednesday, November 16th, 2016): Electron Transport III

Recap: Complex III must have an elaborate structure to conduct the redox reactions required to
eventually donate 1 electron to each cytochrome C.
coQ, which can act as a 1 or 2 electron carrier (because it can exist in a stable 1 electron, radical
state), mediates the division of individual electrons from pairs as theyre run separately through
redox pathways to reach each cytochrome C (via the Q- Cycle).
A total of 4 electrons are derived from molecules of coQH2 per Cycles 1 and 2:
(2) are passed on to the cytochrome Cs: coQH2 ISP cytochrome c1 cytochrome c.
(2) are used to regenerate the semiquinone form of coQ (in Cycle 1) and coQH2 (in Cycle 2) after
theyve been passed through the cytochrome/heme bH and bL pathway.

Complex IV:
IMS

(cytochrome c)

CuA complex: closest to the IMS, it is the first element of Complex IV to be reduced. Two His residues
hold the copper in place (like the pyrroles of cyotchromes)
eHeme a: acts as a redox potential buffer, like cytochrome b does for Complex II.
eHeme a3 + CuB: this complex does the heavy lifting.

Complex IV moves electrons and protons through a redox cycle.

Complex IV moves PROTONS in a way that is completely unique from the specific ways in which
Complexes I and III do so:
Protons are carried like in Complex I: proteins undergo conformational changes, taking protons
from the matrix with sidechains and releasing them into the IMS with other sidechains. These
conformational changes occur because of redox reactions. Protons transported across the IMM in
this fashion are called Vectorial protons: this is a unidirectional pathway.
Protons are also bound to and released by the redox species of Complex IV themselves:
Hemes/cytochromes a and a3, and Cus A and B. Protons transported across the IMM in this
fashion are called Scalar or Chemical protons. Note that the ligands associated with CuA
complex and CuB carry the protons, not the coppers themselves.
The protons transported by Complex I were Vectorial protons; the protons transported by
Complex III were Scalar protons, distributed by coQ. Complex IV can do both, and its
mechanism for transporting chemical protons is unique from Complex IIIs.

Ferric heme irons can bind -OH (hydroxyl) groups well. These hydroxyl groups can act
as proton sources for the release of protons into the IMS.
Different oxidation states for Fe and Cu have different ligands. Different ligands affect
what these Fe and Cu atoms can bind. An FeII-CuB(I) binuclear complex ultimately is
converted into an FeIV-CuB(II) complex as O2 is reduced.
FERROUS iron (Fe2+) in Heme a3 can bind O2 (just like in Hemoglobin).
FeII+O2 is a LIGATION: no redox reactions occur.
FeII+O2 can shift valences to what is essentially FERRYL iron (FeIV)+ a
superoxide anion. At this point, FeIV is bound only to 1 O, the other is bound by
CuB(II).
Ultimately, the oxygen atoms are split up between the Heme a3 Fe and the CuB:
O-FeIV Heme a3 or O-CuB
After the O atoms are each protonated, FERRIC iron (FeI) is bound to OH, and
CuB is bound to OH as well. The FeIV-CuB(II) complex can contribute 3
electrons simultaneously for reduction, compiled after theyre received from
Heme a/CuA complex.
A conserved TYR 244 residue PROVIDES THE 4TH REQUIRED ELECTRON.

In either situation, the oxygen atoms (OH at this point) from each complex are then combined with
protons, generating H2O: where do they come from? The MITOCHONDRIAL MATRIX.
As H2O is formed, it diffuses into either the IMS or the matrix, without specificity.
The formation of H2O in this fashion represents SCALAR proton movement: protons are consumed
FROM the matrix and CHEMICALLY converted into H2O. 2H+ provided via the K-Channel, 2H+
provided via the D-Channel.
Additionally, four VECTORIAL protons are pumped from the matrix into the IMS as O2 is reduced in
coupled reaction. Transported via the D-Channel to the Exit Channel.
8 protons are required to generate 2 molecules of H2O in the cycle employed by Complex IV.

Throughout the ETC, different mechanisms are employed to enhance a proton gradient between the IMS
and the mitochondrial matrix.
Complex I:

proton wire/membrane Bohr Effect: side chains bind and release H+s VECTORIAL

Complex III:

coQ ( a redox actor) carries protons with it and deposits/consumes them. SCALAR

Complex IV:

chemical reactions consume H+ from the mitochondrial matrix and react them with O2
reduction products (-OH) to form H2O. In a coupled reaction, 4 additional protons are
displaced vectorially from the matrix to the IMS
SCALAR & VECTORIAL
****[H+] Matrix < [H+] IMS***
8

This is the gradient that drives ATP production by ATP synthase!

Proton movements at each Complex do NOT follow specific stoichiometric values.

Complex V: the ATP Synthase: the proton gradient established by the ETC is the driving force for
this enzyme. NONE of the ETC complexes generate any ATPs on their own at ANY POINT.
The ATP synthase is composed of many subunits:
Base (F0 subunit): composed of several hydrophobic C-subunits, it is membrane embedded.
Each C-subunit possesses an H+ channel, which work with the proton gradient across the IMM to
initiate ATP synthesis.
Stalk (Gamma chain): connecting F0 to F1, it provides the mechanical driving force to reverse
F1s natural activity as an ATPase. The chain can be proteolytically cleaved, separating F0 and F1.
Head (F1 subunit; consists of 3 - complex subunits): retains its function as an ATPase when
separated from F0 (it hydrolyzes free ATP). It is oriented into the MATRIX.

Matrix

IMS
ATP Synthase produces ATP from ADP + Pi by a Binding-Change Model in 3 Steps.
- pairs form a TRIMER of HOMODIMERS. It is the chain of each dimer which binds ADP or ATP.
Each - complex can sustain three different conformations:

LOOSE

TIGHT

OPEN

1) LOOSE conformation - complex binds ADP + Pi.

2) A concerted conformational change then occurs in all three - complexes:
a. the formerly LOOSE complex, now bound to ADP +Pi, becomes TIGHT.
b. the formerly TIGHT, ATP bound complex becomes OPEN.
c. the formerly OPEN complex becomes LOOSE.
3) The energy involved in these conformational changes converts ADP +Pi in the TIGHT complex
to ATP; ATP is RELEASED from the newly OPEN complex. The newly LOOSE conformation
will bind ADP + Pi and the cycle will repeat itself.
9

So what drives the conformational changes in these - complexes? (Where does the energy come
from?) The amino acid side chains in the 3 - complexes interact with the Gamma chain amino acids.
Each F0 base contains about 10 c-subunits. Each contains a channel which is used to diffuse protons
down their concentration gradient from the IMS to the matrix: this diffusion provides sufficient energy to
turn the c-subunits, move the Gamma stalk, and in turn alter - complex conformations, generating ATP.
Protons make ATP via the transmission of MECHANICAL energy up the Gamma stalk. The -
complexes themselves dont rotate! Only F0 does (in its c-subunits) as protons pass through it.
Interrupting c-subunit turning OR - complex-Gamma stalk interactions will STOP ATP synthesis.
OSCP (Oligomycin Sensitivity Conferring Protein): where oligomycin binds to the ATP synthase,
STOPPING the Gamma stalk from distorting - complexes. Oligomycin is a true ATP synthase inhibitor.
C-subunit proton channels which permit H+ movement are lined with NEGATIVELY CHARGED amino
acid residues (i.e. aspartic acids)

Proton (H+)
Poor

10

Lecture 32 (Friday, November 18th, 2016): Oxidative Phosphorylation

HOW is ATP production related to O2 consumption by Complex IV?
Complexes I, II, and III all drive Complex IV; Complex IV (and how much O2 it takes in) drives ATP
synthase (oxidative phosphorylation)
Recap: Complex IV moves protons with a proton wire/membrane Bohr Effect. The chemical reduction
of O2 to H2O also consumes scalar protons, but these are not the same kind of scalar protons
we see in Complex III: protons in Complex IV are NOT bound to and administered by redox
carriers/actors.
ATP Synthase achieves catalytic activity by an Approximation Mechanism: the enzyme forces
the reactants together (within close proximity) to induce orbital overlap and forced interactions
(also called orbital steering). Conformational changes in the - complexes induced by the
Gamma stalk force ADP and Pi together.
INHIBITION of Oxidative Phosphorylation in the ATP Synthase:
ETC Inhibitors:

Complex IV: cyanide (blocks both coupled and uncoupled respiration with all
substrates)
Complex III: antimycin A (at the Qi binding site), stigmatellin (at the Q0
binding site)
Complex II: thenoyl trifluoroacetone (TTFA)
Complex I: Rotenone, amytal

Phosphorylation Inhibitors inhibit the rotational mechanism of the ATP synthase: Oligomycin
IF1: an endogenous inhibitor of the ATP synthase. Functions to stop the synthase
from retaining its catalytic activity when it cannot or need not be driven by the
ETC (at high ATP concentrations, perhaps).This occurs at LOW/ACIDIC pHs,
when there is little available O2.**
If the synthase isnt driven by an H+ gradient (aka by the ETC), it can quite
easily REVERSE, like when F1 is separated from F0: F1 can act as an ATPase, as
there is no more mechanical driving force to initiate orbital overlapping between
ADP and Pi.
Allowing the synthase to run in reverse as an ATPase would be a waste of ATP.
Uncoupling Agents inhibit no reactions (neither the ATP synthase or the ETC are affected), they instead
abolish the obligatory linkage between the ETC (oxidation) and the ATP synthase (phosphorylation).
They operate by dismantling the H+ GRADIENT across the IMM.
Dintrophenol (DNP): formerly used in shoe polish; linked to weight loss.
FCCP/CCCP: hydrophobic molecules which can be charged or uncharged.
Transport Inhibitors: either prevent the export of ATP across the IMM or the import of raw materials
across the mitochondrial membrane. They inhibit the ATP-ADP translocator.

11

Atracytloside and its derivative carboxyatractyloside (CATR) inhibit the process

only from the EXTERNAL surface of the IMM.
Bongkrekic acid exerts its effects only on the INTERNAL surface of the IMM.
Krebs Cycle Inhibitors: arsenite

Uncouplers, and the P/O Ratio:

ATP synthesis stoichiometry in terms of H+ (how many H+s moved per ATP generated?) is
controversial, and far from constant: regulatory mechanisms lead it to vary extensively.
The argument is made that there are 10 c-subunits in F0, each of which can be rotated a
complete 360 degrees by the passage of one H+ through it (10H+s in total). Therefore, 10H+ are
used to generate 3 ATP: each - complex in F1 can make one ATP from ADP + Pi in a complete
rotation.
IF some ETC components are BYPASSED, less H+ are made available (the H+ gradient is
less contributed to) to turn the F0 c-subunits, and therefore <3ATP are made.
We can consider entering the ETC at different locations and the assumed ATP yield from the ATP
synthase which would result:
@Complex I:

3 ATP

@Complex II: 2 ATP

@Complex III: 2 ATP
@Complex IV: 1 ATP
(Loosening in the coupling mechanism between the ETC and the ATP synthase will vary theis
stoichiometry)
Tetramethyl-p-phenylenediamine (TMPD): donates electrons DIRECTLY to Complex IV; it is
reduced by ascorbic acid (vitamin C). Even if Complexes I, II, and III are deactivated or
inhibited by some means, if we have TMPD we can still make 1 ATP at a time.

The (ATPs generated)-to- (protons moved across the IMM) ratio is related to the number of
oxygen molecules consumed by Complex IV.
If the entire ETC is run leading up to Complex IV, one molecule of O2 will be taken up.
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IMPOSING AN ARTIFICIAL H+ GRADIENT IN THE ABSENCE OF THE ENTIRE ETC

CAN SUCCESSFULLY DRIVE ATP SYNTHASE.

The mechanism for breaking the connection between oxidation (the ETC) and phosphorylation
(the ATP synthase): Uncouplers
ALL uncouplers can cross the IMM easily in either their protonated or deprotonated
forms. This will easily destroy the H+ gradient.

Endogenous uncouplers exist in adipose and muscle tissue: UCP2 and UCP3. Why?
We know that uncouplers are linked to weight-loss phenomena, as observed in
the side-effects of DNP from shoe polish. Why? Uncoupling means that food is
oxidized without ATP generation.
Endogenous uncouplers regulate metabolic rates. Inefficient reactions are so
called because they generate heat. UCP2 and UCP3 make reactions
selectively inefficient to retain/produce heat in the mitochondria and
regulate metabolism/homeostasis.
Again, UCP2 and UCP3 dont react with either the ETC or the ATP synthase.
They dismantle the H+ gradient.
In the presence of an uncoupler, the ETC and the ATP synthase function independently.
Ex: Oligomycin ceases rotation in the synthase, the H+ gradient remains fixed
and builds up, and the resultant protons will provide a force to oppose the ETC.
ATP is not produced, eventually the ETC cannot run, and the Krebs Cycle ceases
to function (succinate dehydrogenase is a part of Complex II).
INHIBITING THE SYNTHASE DESTROYS MITOCHONDRIAL
FUNCTION COMPLETELY UNLESS AN UNCOUPLER IS UTILIZED.
Later, we will discuss the relationship(s) between oxidation and phosphorylation in the mitochondria and
carbohydrate and fatty acid metabolism. Fatty acid metabolism drives the Krebs Cycle: its final product is
acetyl-coA.
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Lecture 33 (Monday, November 21st, 2016): Fatty Acid Degradation I

Obesity: a national
epidemic. 1 in every 3
adults is obese; 17%
of children.
Can cause high blood
pressure, cholesterol,
increased risk of
cardiovascular
disease.
Obesity and Systemic
Inflammation:
dividing adipocytes
release certain factors,
which recruit immune
cells. They also
release proinflammatory factors,
leading to an increase
cancer-risk.
Why do people who lose large amount of weight tend to gain it all back? Our bodies are well adapted to
maintain a narrow weight-range. Homeostatic mechanisms developed evolutionarily over time tend to
bring the biggest losers back to their original weights.
Gut microbiota and obesity: obese individuals possess a different flora than do individuals of a typical
weight. The obese are better at taking energy from food: they derive more short-chain fatty acids from
carbohydrates than do normal individuals.
Classes of Lipids:

Free fatty acids

Triglycerides: glycerol backbone + three fatty acid chains
Cholesterol: 27-carbon compound that possesses an amphiphilic OH at the three
position on its terminal 6-membered ring. This gives the molecule some polarity
and unique properties in hydrophilic/phobic environments.
This OH group can have a fatty acid chain added to it to generate a very
HYDROPHOBIC cholesteryl ester.

- C C COOH OXIDATION takes place at the position on a carboxylic/fatty acid chain.

The x notation is used to describe a fatty acid chain with a double bond x
carbons away from the terminus of the carbon tail.
6 fatty acids: converted to PRO-inflammatory factors. BAD
3 fatty acids: converted to ANTI-inflammatory factors. GOOD
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Corn oils are deficient in 3 fatty acids, and they are a mainstay in the so-called Western diet.
The

FASTING state is associated with high levels of GLUCAGON, and in some

cases EPINEPHRINE.
The FED state is associated with high levels of INSULIN.
Control of Food Intake: How do you know when you are hungry?
Hormones/lipid mediators regulate food intake.
Ghrelin: peptide hormone released from the stomach. Acts on the hypothalamus
to STIMULATE food intake.
Leptin: hormone released by adipose tissue. Affects the hypothalamus to REDUCE feeding. LEPTIN
LEVELS ARE ELEVATED IN OBESE INDIVIDUALS.
Theory of Leptin Resistance: obese individuals become DESENSITIZED to the effects of leptin due to
chronically ELEVATED levels.
NO Leptin expression leads to obesity; TOO MUCH Leptin expression leads to obesity.

1) Fatty acid metabolism occurs primarily in the SMALL INTESTINE:

Triglycerides: hydrophobic; Y-shaped, with hydrophobic tails articulating with the environment. They
pack into globules in polar solutions.
Pancreatic Lipase
Monoglycerides + (2) Free Fatty Acids
15

+Cholesterol
to small intestinal epithelial cells.
Triglyceride globules are emulsified by bile salts from the gall bladder. Bile acids have cholesterol-like
structures, with conserved charged attachments: they act as detergents. They increase the total surface
area on which pancreatic lipase can act.
Pancreatic lipase cleaves a triglyceride at the 1 and 3 positions, liberating the 1 st and 3rd fatty acids from
the glycerol head-group.
2) Chylomicron Synthesis: for the transport of triglycerides within the bloodstream.
Monoglyceride and the two Freed Fatty Acids are recombined to regenerate a
Triglyceride inside small intestinal epithelial cells.
Cholesterol molecules are added to acyl chains to generate hydrophobic cholesteryl
esters.
These triglycerides and cholesteryl esters are packaged together beneath a phospholipid
monolayer into CHYLOMICRONS.
Some cholesterol molecules are embedded in the membrane, with their
amphiphilic OH groups pointing outward.
Chylomicrons are released from the intestinal epithelium into the lymph and then into the
blood.
3) In the blood, Lipoprotein Lipase attacks chylomicrons and re-divides triglyceride into a
Monoglyceride and two Free Fatty Acids at the capillary beds of adipose and muscle tissues.
The enzymes cleaving activity is very similar to that of pancreatic lipase.
MAG + (2)FFAs are taken into MUSCLE cells for ENERGY; they are taken into
ADIPOSE tissue for STORAGE (TAGs are resynthesized, again).
What if youre missing lipoprotein lipase? Fat accumulates in the blood in the form of
chylomicrons.
Alipogene Tiparvovec (Glybera): viruses that can express Lipoprotein Lipase are
injected into muscle tissue.
4) In adipocytes, dietary lipids are repackaged into lipid droplets for storage. They are very similar to
chylomicrons.
When energy is desperately needed, these droplets can be broken down and free fatty
acids can be resynthesized for energy.
Lipid droplets are dynamic: they grow and shrink based on the rate of lipid turnover.
Fasting State: elevated glucagon levels stimulate LIPOLYSIS: the hydrolysis of lipid
droplets. Epinephrine also stimulates lipid liberation as a part of the fight-or-flight
response.

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ATGL: Adipocyte Triglyceride Lipase: cleaves off a free fatty acid from the 1 position.
HSL: Hormone Sensitive Lipase: cleaves off a free fatty acid from the 3 position.
MAGL: Monoacylglycerol Lipase: cleaves the glycerol head group from the final free
fatty acid.
What is a consequence of ATGL dysfunction? Obesity!
ATGL deletions lead to enlarged, fatty hearts in mice, with shortened life expectancies.

How can we regulate lipolysis?

Lipolysis normally
occurs at a low basal
rate.
At low cellular
energy levels,
glucagon and
epinephrine bind
their extracellular
receptors.
This activates
protein kinase A.
PKA
phosphorylates
perilipin1.
Normally, perilipin1
sequesters ABHD5,
but when
phosphorylated,
perilipin1
dissociates from ABHD5. ABHD5 then has the opportunity to recruit ATGL to the lipid droplet
surface while upregulating its activity.
Chanarin-Dorfman Syndrome: mutations resulting in nonfunctional ABHD5. Enlarged
LIVER (hepatomegaly) results. TAGs cannot be broken down, and therefore they build
up in tissue.
PKA also phosphorylates HSL, recruiting it to the lipid droplet.
MAGL is not regulated: it is a garbage collector, which finishes breaking down MAG.
The free glycerol head group of the TAG released can be used for gluconeogenesis in some cases.
17

18

Lecture 34 (Monday, November 28th, 2016): Fatty Acid Degradation II

5) Free Fatty Acids generated by lipolysis in adipocytes are released into the bloodstream and mix with
albumin.
Upon reaching their target tissues, the acids are broken down via -Oxidation for energy. Free fatty
acids are converted within the mitochondrial matrix into ACETYL-COA.
Long chain fatty acids must
first be transported into the
mitochondrion.
Acyl-CoA synthetase
transforms fatty acids into
Acyl-CoAs in a two-step
mechanism.
The energetic equivalent of 2
ATPs is consumed: this is an
energetically costly process.
The acyl-CoAs must be
converted into acyl-carnitines
to cross the mitochondrial
membrane.
CPT1: converts acyl-CoA
into acyl-carnitine. Located
in the OMM.
CPT2: converts acyl-carnitine back to acyl-CoA and exports free carnitines back into the IMS. Located in
the IMM.
CPT 1 and 2 together are considered as the Carnitine Acyltransferase Complex.

19

6) Once Acyl-CoA has been regenerated within the mitochondrial matrix, -Oxidation can occur,
producing:
1 Acetyl-CoA, 1 FADH2 (Step 1), 1 NADH (Step 3) per round.
1. Acyl-CoA Dehydrogenase (AD): abstracts 2Hs from the acyl-CoA: one from the C and the
other from the C. A trans - double bond results.
2Hs are given to FAD in this step, generating 1 FADH2.
There are multiple isoforms of this enzyme for different chain lengths.

2. Enoyl-CoA Hydratase (EH): - trans double bond generated in step 1 reacts with H2O, which
is added to the double bond as an H on the C and an OH on the C.
3. HAD: abstracts 2Hs, creating a -ketone and generating one molecule of NADH.
4. Thiolase (KT): A C-C bond cleavage occurs at the KT site. coA attacks the C.
Acetyl-CoA leaves; a new molecule of coA has attached to the C of the fatty acyl-CoA chain
(which is now two carbons SHORTER). The chain returns to Step 1 and runs through the
cycle over and over, cleaving off two carbons at a time.

The generated Acetyl-CoA is then shuttled to the Krebs Cycle, feeding

it and reinforcing oxidative phosphorylation while decreasing
metabolic glucose-dependence.

20

21

Ex: 16-carbon palmitate undergoes seven rounds of -oxidation before it is broken down completely into
8 molecules of Acetyl-CoA. The energetics:

FADH2

10.5 ATP equivalence/molecule

NADH

17.5 ATP equivalence/molecule

Acetyl-CoA

10 ATP equivalence/molecule

-2 ATP per fatty acid to generate Acyl-CoA intermediate

106 total ATP equivalence generated per molecule of palmitate

The -oxidation of UNSATURATED fatty acids is extra problematic

for Enol-CoA Hydratase (EH).

Cells have evolved pathways to maintain EH

function when oxidizing unsaturated fatty acids.

Eventually, EH is faced with a - double bond in the substrate.

Enoyl-CoA Isomerase: shifts the - double bond

into the - position. This allows -oxidation to
continue.

Double bonds further up the chain become

problematic when they (eventually) reach the 4,5
position to the carbonyl: they prevent the fatty
acyl-CoA chain from being used as a substrate by
EH.

Dienoyl-CoA Reductase: turns an - double bond

and a 4,5 double bond into a single - double bond.
At this point, the Enoyl-CoA Isomerase can shift this
bond into the - position, and -oxidation can run to completion.

-Oxidation for ODD-chain fatty acids: the final product will be Propionyl-CoA ( a three carbon fatty
acid, not Acetyl-CoA; not large enough to be a substrate for AD).
22

Propionyl-CoA can be converted to succinyl-CoA and passed onto the Krebs

Cycle.
7) The BRAIN does NOT use fatty acids for energy, because it cannot take them up.
Acetyl-CoA is converted in the LIVER into Ketone Bodies, which are sent via the bloodstream to target
tissues (like the brain) where they are reconverted into Acetyl-CoA and used for -oxidation.
Fatty acids cannot readily diffuse across the blood-brain barrier. Alternate energy options during fasting
include glucose (regenerated from glycogen stores), triglycerides, proteins, and ketone bodies, which are
produced in starvation.
The use of ketone bodies spares/maintains glucose stores for the brain.
Ketone Bodies are essentially 4 carbon units, formed from the fusion of 2 Acetyl-CoAs without their
CoAs attached.
They are formed when the blood concentration of fatty acids is high: both in individuals with a high fat
diet and in individuals who are starving.
The products of -oxidation INHIBIT glycolysis in the liver, REDUCING glucose reliance and
maintaining glycogen stores.
Ketogenesis is like -oxidation run in reverse:
HMG-CoA Lyase is a LIVER-SPECIFIC
ENZYME: ketone bodies are generated
exclusively in the liver before being distributed
via the bloodstream to other tissues. The lyase is
found ONLY in the mitochondira!
The Acetyl-CoA generated with each Ketone
Body remains in the liver.
Ketone Bodies are often sent to the HEART and
the BRAIN.
Upon reaching their targets, the Ketone Bodies
are reconverted to Acetoacetyl-CoA and then to
Acetyl-CoA by thiolase. They successfully:

Provide energy to other tissues

Transport Acetyl-CoA from the liver to
tissues
Lower glucose demand to support the
brain in starvation

EXCESS Ketone Bodies induces

KETOACIDOSIS in two cases:
Diabetic: insulin disallows glucose uptake, and the body believes it is starving.
Alcoholic
23

Lecture 35 (Wednesday, November 30th, 2016): Fatty Acid Biosynthesis

Acetyl-CoA is
transported from
the matrix to the
cytosol as
CITRATE; it is
converted back to
acetyl/malonylCoA and is used to
generate long
chain fatty acids.
Fatty acid
biosynthesis runs
counter to oxidation, and
they are
mechanistically
very similar. They
represent each other, in reverse.
COMPARTMENTALIZATION of oxidation in the mitochondrion and biosynthesis in the cytosol allows
for the co-regulation of the two pathways with respect to one another.
-oxidation generates a LOT of energy; fatty acid biosynthesis is energetically costly.

The Fed State: characterized by high INSULIN levels: by raising systemic insulin levels, we stimulate
fatty acid biosynthesis and thus ensure that fatty acids can be stored for future use in a Fasting State.

Tricarboxylic Transport System:

Acetyl-CoA is produced in the mitochondrial matrix by -oxidation. It can then be
coupled with oxaloacetate to produce CITRATE, which can be transported into the
cytosol.
Acetyl-CoA and OAA are immediately regenerated from citrate upon entrance into the
cytosol.
From there, OAA malate pyruvate imported back into the matrix. NADPH is
produced when malate is converted to pyruvate.

24

Overall Reaction of Fatty Acid Biosynthesis: 2-carbon units (Acetyl/Malonyl-CoA) are conjugated
over and over to produce Palmitate (a 16-carbon fatty acid).

1. The COMMITTED STEP: Acetyl-CoA Carboxylase uses ATP and a biotin functional group to
convert Acetyl-CoA into Malonyl-CoA by adding a CO2 from bicarbonate:
Acetyl-CoA + HCO3- +ATP Malonyl-CoA + ADP +Pi
Large, impressive enzyme:

BC site: where CO2 attaches to biotin

CT site: catches the CO2-bound biotin, which transfers
the CO2 to Acetyl-CoA to make Malonyl-CoA.

Malonyl-CoA INHIBITS -oxidation by acting to inhibit CPT1.

2. In a complex, multi-step process, Acetyl-CoA and Malonyl-CoA come together within Fatty
Acid Synthase (FAS) to generate Palmitate after 7 cycles:
Two carbons are added to the growing chain PER CYCLE in the form (originally) of an
Acetyl-CoA.
Acyl Carrier Protein
(ACP): structurally
analogous to coA. Holds
growing fatty acid chain in
place so it cannot escape
the enzyme; also delivers
the chain to each
enzymatic subunit
sequentially. The ACP
remains tethered to the
future carboxylic carbon
of the growing fatty acid.
No intermediates can
escape, and the catalytic
rate increases for it.
Acetyl-CoA enters the
enzyme at the MAT site,
where the Acetyl group is
transferred to the ACP.
The ACP delivers the
acetyl group to the KS site,
where it binds to the
enzyme itself. The ACP
then returns to the MAT
25

site, latches onto Malonyl-CoA, and brings it to the KS site to meet the enzyme-bound
Acetyl-CoA.
Here, CO2 is released as the Malonyl-CoA and Acetyl-CoA are conjugated together,
forming a 4-carbon unit still bound to the ACP, called Butyryl.
The end product of the first fatty acid synthase cycle (butyryl-ACP) swings back to
the KS active site and becomes tethered there, again releasing ACP and allowing the
ACP to acquire another malonyl group for further addition onto the growing fatty acid
chain. These cycles are repeated until palmitate is produced and released from the
enzyme.
The entire process costs 7 ATP:
one for each cycle, because
converting Acetyl-CoA into
Malonyl-CoA requires 1 ATP.
VERY COSTLY!
Fatty Acid Biosynthesis will
therefore only occur when both
ATP and Acetyl-CoA
concentrations are HIGH!
Notes:
Palmitate can be expanded into
longer fatty acid
chains.
Densaturases can act upon these to
form double bonds.
Mammals CANNOT de-saturate past carbon 9.

Acetyl-CoA Carboxylase is STIMULATED by INSULIN (in high energy states) and INHIBITED by
GLUCAGON and EPINEPHRINE (in low energy states).

AMP-Activated Protein Kinase (AMPK): activated when ATP levels are LOW and AMP levels are
HIGH.
AMPK phosphorylates Acetyl-CoA Carboxylase, deactivating it.
This insures that fatty acid biosynthesis will only occur when the cell can afford to
carry it out, not in low energy states.
Fatty Acid Synthase is upregulated in cancer cells: cells need fatty acids for energy and to grow and
divide.
Tumors SHRINK under the pressure of a FAS inhibitor.

26

Acetyl-CoA Carboxylase is also upregulated in cancer cells.

Bioactive Lipids: in addition to providing energy for cellular metabolic needs, fatty acids are also
converted into potent signaling molecules.
Prostaglandins:
Cyclooxygenases:

COX1: expressed in most tissue

COX 2: induced during inflammation. Converts arachidonic acid
into PGH2, which can be used to generate a variety of
prostaglandins. COX2 is upregulated in cancers; low dose
aspirin reduces the extent of the metastatic potentials for some
cancers.

Prostaglandin E2 (PGE2):

produced by PGE synthase from PGH2

Central mediator of the fever: cytokines (IL1, IL6,
TNFa) induce the expression of COX2 in the
endothelium lining the brain.
PGE2 turns up the hypothalamic thermostat and
sensitizes pain pathways.
PGE2 reduces appetite by INHIBITING Ghrelin.

Prostaglandin D2 (PGD2): makes you drowsy when youre sick.

COX Inhibitors like Aspirin, Ibuprofen, and Acetaminophen all inhibit PGH2 synthase. Ararchidonic acid
cannot be turned into PGH2, and many metabolites cannot be produced (i.e. PGE2).
COX inhibitors cause gastric ulcers as a side effect. Why? COX1 compounds protect
the stomach mucosa.
Selective COX2 inhibitors were produced and took advantage of a larger active site on
COX2.
Rofecoxib (Vioxx): side effects included heart attacks
Celecoxib (Celebrex): still approved today for severe pain ONLY.
WHY would Vioxx cause heart attacks?
COX1 makes Thromboxane A2 (TXA2), which stimulates platelet aggregation.
COX2 makes Prostacyclin (PGI2), which INHIBITS platelet aggregation.
Selective COX2 inhibitors (i.e. Vioxx) could throw off this balance: clots develop
in the absence of COX2 and break free, heading to the brain or heart and causing
stroke or MI.
27

Leukotrienes:

Lipoxygenase products made from arachidonic acid

Slow-reacting substance of anaphylaxis: Leukotriene C4
Bronchial smooth muscle contraction leads to bronchoconstriction

Resolvins:

mostly anti-inflammatory, synthesized from 3 fatty acids.

Lecture 36 (Friday, December 2nd, 2016): Cholesterol Metabolism

Sources of Cholesterol:
Endogenous: the LIVER makes
about 1 gram a day (70% of the
daily-required value). This is the
type of cholesterol most
effectively targeted by the best
cholesterol medications.
Exogenous: dietary sources.
Derivatives of Cholesterol:
Cortisol, aldosterone, estradiol,
testosterone, and Bile Salts: the
detergent-like substance generated in the gall bladder to aid in the digestion of lipids. These salts
represent the only known mechanism for cholesterol excretion from the body.

Cholesterol Biosynthesis:
Occurs predominantly in the liver
Synthetic enzymes are found in the CYTOSOL (just like FAS). Why? Compartmentalization allows for
the co-regulation of catabolic and anabolic pathways simultaneously.
Acetyl-CoA is the major carbon donor.
Energetically costly: occurs only in the Fed State.
The Tricarboxylic Transport System transports Acetyl-CoA as CITRATE from the matrix into the
cytosol, where it can be used to generate cholesterol. Acetyl-CoA is converted to ISOPRENE UNITS in
order to be viable in this synthesis.
HMG-CoA will be made from 3 molecules of Acetyl-CoA by cytosolic enzyme similar to those for
Ketone Body Synthesis. HMG-CoA Lyase is NOT found in the liver cell CYTOSOL. In the cytosol,
HMG-CoA is reserved purely for cholesterol biosynthesis.
28

Four Stages:
1. Acetyl-CoA converted to HMG-CoA by HMG-CoA Synthase; HMG-CoA converted to
isopentenyl-pyrophosphate by HMG-CoA Reductase in a 4-step mechanism that requires 2
NADPH and 3 ATP and represents the REGULATORY STEP:
a. HMG-CoA Reductase: 4 electrons used to reduce the thioester to an alcohol.
Mevalonate is produced, and the 2 NADPH are used.
b. Mevalonate-5-Phosphotransferase: the terminal phosphate from 1 ATP is added to
mevalonate to produced Phosphomevalonate.
c. Phosphomevalonate Kinase: 1 ATP is used to add another phosphate to
Phosphomevalonate, yielding 5-Pyrophosphomevalonate.
d. Pyrophosphomevalonate Decarboxylase: 5-Pyrophosphomevalonate is decarboxylated,
using the 3rd and final ATP, to produce isopentenyl pyrophosphate.
2. Condensation of 6 isopentenyl pyrophosphates to make squalene (a 30 carbon compound):
a. Dimethyl allyl pyrophosphate (5 carbons) is produced from isopentenyl pyrophosphate. It
is conjugated with a molecule of isopentenyl pyrophosphate; this product is then
conjugated with another 5 carbon group to generate a 15 carbon product.
b. (6) 5-carbon units come together in total when (2) 15-carbon pyrophosphates are
conjugated by squalene synthase to produce squalene.
3. Cyclization of Squalene to make Lanosterol (the terminal precursor to cholesterol): in the
presence of O2, squalene epoxidase attaches one oxygen to a single carbon-carbon double
bond using 1 NADPH. The resultant product is 2,3 oxidosqualene.
a. Oxidosqualene cyclase reaction: the enzyme-mediated protonation of the epoxide.
b. When the epoxide ring opens, an electron deficient center is left behind.

4. Modifications to Lanosterol make Cholesterol: one reduction, 3 methyls removed.

29

How is cholesterol biosynthesis regulated?

HMG-CoA Reductase is a KEY STEP, and it is highly regulated. Leads to the creation of
isopentenyl pyrophosphate.
In times of need, the reductase is phosphorylated and inactivated by
AMPK: why? The cell cannot afford to make cholesterol and spend ATP
when energy is low.
SREBP: a protein retained in the ER of cells when cholesterol levels are high/normal. When cholesterol
levels are LOW, SREBP is freed from the ER and heads to the Golgi.
Two proteases, Sites 1 and 2, sequentially cleave SREBP, first in half and
then again.
After the second cleavage, a small, soluble portion is freed and
proceeds to the nucleus, where it induces the transcription of mRNA
for HMG-CoA reductase.

How is cholesterol transported to target tissues?

It is absorbed from the diet in the small intestinal epithelial. Acyl-CoA turns cholesterol into hydrophobic
cholesteryl esters, packages them into chylomicrons, etc.
In the liver, cholesterol is converted into cholesteryl stearate (an ester).
These cholesteryl esters/free cholesterols are packaged into VLDLs (Very
Low Density Liver Proteins), lipoproteins that are released into the
bloodstream.
Cholesterols are confined to the phospholipid monolayer of the VLDL
while the cholesteryl esters are packed into the hydrophobic interior.
The Apolioprotein B-100 wraps itself around the VLDLs, which are
much smaller than chylomicrons.

30

The liver takes up cholesterol-rich chylomicrons, repackaging their cholesterol/cholesteryl esters

with its endogenous cholesteryl esters into VLDLs, which are released and acted upon by
lipoprotein lipase.
Cholesterol-rich VLDLs become LDLS when they lose their
triglycerides to tissues after exposure to lipoprotein lipase.

The Blood-Brain-Barrier prevents the brain from conducting too much cholesterol metabolism at all.
HDLs transport excess cholesterol back to the liver, too, for disposal.
Peripheral tissues have receptors for B-100, which allows cells to take up LDLs.
LDL is called Bad Cholesterol because it can often be deposited in arteries in excess as it
attempts to return back to the liver after exposure to lipoprotein lipase.
The more cholesterol one has in his or her blood, the more likely that person is to develop
atherosclerosis and Coronary Artery Disease.
LDL particles can be oxidized; they stick to endothelial vessel linings and remain there.
They send signals to macrophages, who come and ingest them, turning into foam cells
31

and in turn signaling for more macrophages, which eventually leads to calcium deposits
in arteries which can break free, head for the heart or brain, and cause a MI or a stroke.
HDL is called Good cholesterol because it is cholesterol being transported back to the liver
(unidirectional) in order to be excreted via bile salts.

The best drugs designed to lower cholesterol target endogenous cholesterol: STATINS:
Statins INHIBIT HMG-CoA Reductase, lowering cellular cholesterol concentration, causing
the induction of HMG-CoA Reductase and LDL receptors, lowering serum cholesterol
concentration through an increased liver cellular uptake of LDLs.
Zetia inhibits intestinal cholesterol absorption (it targets dietary cholesterol). It is less effective
than statins are.

32

Lecture 37 (Monday, December 5th, 2016): Metabolic Regulation I

ANABOLIC reactions are generally confined to the CYTOSOL;
CATABOLIC reactions are generally confined to the MITOCHONDRION, typically.
In cancer cells, however, the Krebs Cycle (in the mitochondrion) will run in reverse (ANABOLICALLY).

How do the cytosol and the mitochondrion communicate?

Weve discussed proton movement, and the Malate-Aspartate Shuttle for gluconeogenesis from Krebs
Cycle intermediates, which must necessarily be exported into the cytosol in order to reproduce G6P:
Oxaloacetate is converted backwards to malate, which can then exit the mitochondrion through
the IMM, to be reconverted to OAA in the cytosol. There, it is acted upon by
Phosphoenolpyruvate Carboxykinase to regenerate Phosphoenolpyruvate and initiate
gluconeogenesis.
This shuttling of OAA across the membrane disguised as malate also serves to move NADH
produced in glycolysis between the cytosol and the mitochondrion.
Lactic fermentation can generate the NAD+ required for glycolysis.

Glycerophosphate Shuttle: allows for the regeneration of NAD+ and the reduction of FAD to
FADH2 in the IMM conducted by Flavoprotein Dehydrogenase, which acts on 3-Phosphoglycerol.
Eating too much fructose can be dangerous because it allows for the unregulated production of
Dihydroxyacetone phosphate (DHAP) and Glyceraldehyde-3-Phosphate (GAP).
Why? PFK, the major
regulatory step in the
glycolytic pathway, is
bypassed.
DHAP can be shunted
from the glycolytic
pathway and used to
regenerate NAD+ by 3Phosphoglycerol
Dehydrogenase, a
cytosolic enzyme.
The resultant 3Phosphoglycerol can be
oxidized by the
Flavoprotein
Dehydrogenase to

33

regenerate DHAP and FADH2, which is passed along to Complex II of the ETC: succinate
dehydrogenase.
Generating 3-Phosphoglycerol is also important because it can play a role in the cytosolic generation of
triglycerides, which can be metabolized into free fatty acids and eventually oxidized to Acetyl-CoA to fuel
the Krebs Cycle.

The Coordinated Control of Glycolysis and the Krebs Cycle:

Glycolytic Activators: PFK

AMP
Fructose-2,6-Bisphosphate (generated by PFK2)
Fructose-1,6-Bisphosphate: an allosterically enhanced substrate for
PFK in conjunction with ATP, F16BP can upregulate GHAP and 3PG
production by upregulating the function of its own creator.

Glycolytic Inhibitors:

PFK

ATP

Less-potent regulatory steps along the glycolytic pathway:

Feedback inhibition of hexokinase (HK) by G6P.
Pyruvate Kinase (PK) can use its N2 variant not to convert PEP to pyruvate
but instead to phosphorylate glycolytic intermediates 2PG and 3PG, using
PEP as an energy source.

Glycolytic reactions are less-than-regulated by the redox state around them (i.e. [NADH]/[NAD+]),
although to a certain extent, as weve seen, Glyceraldehyde-3-Phosphate is sensitive to NAD+ levels.

Entry into the Krebs Cycle requires the Pyruvate Dehyrogenase (PD) enzyme complex, which is
challenged not to move quickly forward, for the PD complex as the choice to either convert pyruvate to
Acetyl-CoA to feed the Krebs Cycle or to reconvert pyruvate to PEP and initiate gluconeogenesis.
Regardless, conversion of pyruvate to Acetyl-CoA represents a major rate-determining step for the
Krebs Cycle.
The rate-limiting enzyme of the Krebs Cycle is Isocitrate Dehydrogenase, which is ACTIVATED
by Ca2+ and ADP and INHIBITED by a HIGH [NADH]/[NAD+] ratio (high NADH
concentrations).
The purpose of the ETC is to generate NAD+: when the mitochondrion isnt functioning
adequately (like under HYPOXIC conditions), NADH accumulates (the ETC cannot run
efficiently), the ratio aforementioned INCREASES, and the Krebs Cycle enzymes are inhibited,
for -Ketoglutarate Dehydrogenase and Malate Dehydrogenase are also inhibited by this high
ratio.
34

Ca2+ also activates -Ketoglutarate Dehydrogenase (see following graphic).

Regulation depends
on the purpose of a
given pathway at a
certain point in time.
The purpose of the
Pyruvate
Dehydrogenase (PD)
complex can be twofold:
Anabolic:
generate amino acids,
hemes, etc., from
Krebs Cycle
intermediates.
This anabolic
activity is
controlled by
the levels of
the given
anabolites.
Catabolic:
produce NADH and FADH2 for ATP production.
This catabolic activity is controlled by the energy state of the cell.
*** The regulation of the PD Complex at any point in time towards either its anabolic or catabolic
functionality is a big determinant as to where the Krebs Cycle is going to get its Acetyl-CoA from, either
PD or fatty acid metabolism.
When the E1 complex of PD is phosphorylated, the complex is significantly affected.
High NADH concentrations will slow down the PD: no more NADH need be made.

Pyruvate Dehydrogenase Kinase is highly regulated by various affectors: it

PHOSPHORYLATES the E1 complex, DEACTIVATING it.
35

AcetylCoA produced by fatty acid metabolism ACTIVATES the PDK, shutting

down PD: no Acetyl-CoA is required for the Krebs Cycle because there is
already a lot of it.
Pyruvate Dehydrogenase Phosphotase cleaves a phosphate from the
phosphorylated E1 complex.
PDP is activated by INSULIN and the HIGH-energy state, when
fatty acids are less likely to be metabolized for quick-energy:
glucagon and epinephrine stimulate -oxidation of free fatty
acids in the Fasting State.
In order to convert glucose into fatty acids for (eventual) storage in a
high-energy, Fed State, the PD must be active: Krebs Cycle
intermediates are required to generate free fatty acids.

The HEART represents an exemplary catabolic organ. Atherosclerosis can be

determental to its functionality as such.
Under NORMOXIC
conditions:
Glucose and lactate are
metabolize to pyruvate and
utilized to generate AcetylCoA for the Krebs Cycle.
PD is turned OFF (inhibited)
by high levels of Acetyl-CoA
produced by the -oxidation
of fatty acids (the heart
tissue prefers to catabolize
fatty acids for energy).
NADH produced by
extensive -oxidation also
inhibits PD while fueling the
ETC.

Under HYPOXIC conditions:

36

The ETC operates sluggishly,

and the conversion of NADH
to NAD+ is inefficient.
NADH accumulates in
EXCESS, and with the
continued production of
Acetyl-CoA by -oxidation,
PDP is severely INHIBITED,
and the PDK is excessively
activated, making PD activity
nearly neglibile, and
reinforcing the sole
dependence upon -oxidation for Acetyl-CoA by the heart tissue. This may
not be sustainable, and can be stressful (Think: were talking about total
O2 dependence in HYPOXIC tissue. It will not add up!).
In order for the tissue to return to at least a partial dependence upon glycolysis for
Acetyl-CoA (anaerobic metabolism) in ishemic tissue, this almost total inhibition of
PD at E1 must be relieved.
The solution: inhibit the -oxidation of fatty acids: this will reduce Acetyl-CoA and
NADH levels.
Trimemetazidine: prevents the myocardium from using fatty acids for
Acetyl-CoA synthesis and forces it to use glyolysis.
How does it do this? By inhiting the CPT1 pathway: fatty acids cannot be
transported into the mitochondrion for -oxidation. It also accomplishes the
inhibtion of Keto-Acyl-CoA as well, which is the final step in the oxidation
pathway and reduces its success substantially. Any step in the cycle can act
as a regulatory step.

37

Effects of the Fatty Acid Synthases (FAS) overexpression in cancer cells:

Extensive studies have proven that the inhibition of the proliferating cancer cells is possible via FAS
inhibitors.
Orlistat (Xenical) inhibits the gastric and pancreatic lipases, leading to weight loss, in turn shutting-down
FAS.
AMPKs sustained activation INHIBITS tumor growth. Its phosphorylation cascade MEDIATES
catabolic pathways while inhibiting anabolic ones:
ACTIVATES glycolysis by activating PFK-2, which in turn makes the F-2,6-BP that activates
PFK-1.
INHIBITS acetyl CoA carboxylase and thereby inhibits fatty acid biosynthesis, including
lowering levels of malonyl CoA.
Lower malonyl CoA levels relieve inhibition of carnitine acyl transferase, enhancing fatty
acid entry into the mitochondria and subsequent breakdown to acetyl CoA.
Lecture 38 (Wednesday, December 7th, 2016): Metabolic Regulation II

38

Activated-AMPK-

induced catabolic pathways inhibit carbohydrate and fatty acid synthesis reactions
(gluconeogenesis, glycogen synthesis; Fatty Acid Synthase)
AMPK is activated when it is phosphorylated by Liver Kinase B1 (LKB1).
Mutations in LKB1 increase the propensity to develop cancers.
AICAR (aminoimidazole carboxamide riboside) is an endogenous activator of AMPK that triggers cell
death in leukemia cancer cells.
The theme of AMPK: BREAKDOWN. It even inhibits insulin synthesis and secretion in pancreatic cells.

Protein Kinase A: activated by cAMP

PKA in its inactive state is a heterotetramer
composed of 2 regulatory subunits (red) and 2
catalytic subunits (blue).
4cAMP are required in order to bind and remove the
2 regulatory subunits, activating the catalytic
elements of the kinase.

PKA phosphorylates phosphorylase kinase, which later phosphorylates phosphorylase.

PKA carries a natural, built-in, 20-peptide inhibitor that remains associated with its catalytic subunit until
it is activated, at which point the inhibitory piece falls off.

39

Phosphorylase Kinase has a structure which is nearly superimposable with PKAs.

PK does not possess the aforementioned inhibitory subunit, though.

Glycogen breakdown lies at the bottom of the

PKA PK phosphorylation cascade.
(Glycogen) Phosphorylase is activated
both ALLOSTERICALLY by AMP (and
inhibited by ATP) and by COVALENT
MODULATION via phosphorylation by
PKA.

MAXIMUM activation occurs when

phosphorylase is both AMP bound and
phosphorylated, and MIMIMAL activation
occurs when phosphorylase is bound to
little/no AMP and minimally phosphorylated.

40

G-Protein coupled receptors make use of trimeric G-proteins because of the GTPhydrolysis associated with them.
G-Proteins dont phosphorylate downstream target; GTPase activity by the Gproteins REGULATES their activating or inhibiting abilites downstream.
The most important target of G-proteins: Adenylate Cyclase, which
produces cAMP.
cAMP can be destroyed by phosphodiesterase, which turns it into AMP. This enzyme is
not highly regulated, and can have dramatic consequences (i.e. Viagra is a
phosphodiesterase activator).

Adenylate Cyclase:
Integral membrane subunits anchor it
to the cell membrane
Its working end faces into the cytosol
4 subunits: C2b, C2a, C1a, C1b
These subunits can respond to
potent activators: G-proteins
They can also respond to potent
INHIBITORS: Ca2+,
elevated PKA concentrations
(enough active PKA means
that there is sufficient cAMP)

Hormones (epinephrine, etc.)

G-protein coupled receptor + G-protein

Adenylate Cyclase

4 cyclic AMP
phosphorylase

PKA

PK

Glycogen

phosphodiesterase
4AMP
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Lecture 39 (Friday, December 9th, 2016): Metabolic Regulation III

PKA cant do anything until it is activated. Activity arises when the catalytic subunits can separate from
cAMP-bound regulatory subunits, losing their auto-inhibitory subunits in the process.
Adenylate Cyclase produces cAMP; it is stimulated to do so when G-proteins attach to its basal subunits.
G-proteins bind GTP and retain GDP after a hydrolytic step.
The role of GTP is to induce a conformation change in the G-Protein (it doesnt phosphorylate anything)
G-proteins are intimately associated with G-protein coupled receptors.
Receptors transmit signals into the cell via G-proteins after binding extracellular signal molecules.
7 membrane-spanning helices in each G-protein coupled receptor undergo conformational
changes when ligand-bound.
Heterotrimeric G-proteins have 3 subunits:
The -subunit is the largest of the three and pulls most of the weight in signal transduction.
The and subunits are intimately associated (but NOT covalently linked) and separate from the
-subunit when a fresh GTP binds to it.
The GTP-bound -subunit articulates with the adenylate cyclase.
1. The receptor binding to the ligand triggers the -subunit
to surrender GDP and bind a fresh GTP molecule,
shedding the and subunits, which remain intimately
linked.
2. The -subunit has a GTPase activity: after the GTPbound -subunit stimulates the adenylate cyclase, the subunit cleaves the GTP to regenerate GDP as it departs
from the adenylate cyclase.
3. As this occurs, the -subunits affinity for the and
subunits returns, and the heterotrimer reassembles.
4. Before the cycle can repeat, the GDP bound to the
heterotrimer is replaced with a new GTP, which
reinitiates the process if the receptor is ligand-bound.
The adenylate cyclase has a binding site for the - pair
on the C2a subunit which is not potent in activating it.
The C2a -subunit binding site will induce activation
when the -subunit is bound.

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The C1a subunit has a binding site for the -subunit of a different G-protein, one which
serves to INHIBIT, or DEACTIVATE, the adenylate cyclase.

The two different activating/deactivating G-proteins are associated with different receptors and different
GTPase inhibitors:
Cholera Toxin inactivates the GTPase capabilities of the -subunit of the AC-Activating Gprotein.
Treatment: replenish the patient with excess water and salts to restore ion concentrations depleted
by ion channels forced to stay open: when the GTP-bound -subunit cannot convert its GTP to
GDP, it cannot turn off the cascade it initiates, and so AC is continuously stimulated to produce
cAMP and carry out its associated cascades.
One of these cascades involves phosphorylating (and thus opening) a Cl- anion transport channel
in the cell membrane.

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Pertussis Toxin inactivates the GTPase capabilities of the -subunit of the AC-Deactivating
G-protein.
MAP Kinase Cascade: important for inflammatory responses; similar in process and
expansiveness to the phosphorylase cascade.

Anthrax targets these MAP Kinase pathwats, particularly at the level of the MEKs.
Lethal factor cleaves the MEKs, shutting down the cascade, and rendering the body
incapable of initiating an inflmmatory response as the Bacillus which generates these
toxins is free to replicate in the immunocomromised host. Bacteremia and death result.
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Healthy, this pathway mounts a cytokine-rich inflammtion response.

Tyrosine Kinases are frequently associated with signal transduction in cancers (i.e. Src).
They employ auto-activated pathways triggered by the binding of signals to Src.
Gleevec (Imatinib): the earliest Tyrosine kinase inhibitor developed; very effective in
CML patients.

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