Study exercise Biology 13 15

1. How are the two products of glycolysis connected to the reactions

of the TCA cycle?
2 products of glycolysis Pyruvate & NADH, can be metabolized in 2 different ways depending on
cell type in which they are formed & the presence or absence of oxygen

In presence of O2 in mitochondria, aerobic organisms extract large amounts of energy from

the pyruvate& NADH made during glycolysis (>30 more ATPs)
PYRYVATE
o Used as a generator of TCA cycle
In the mitochondrion, several enzymes catalyze the conversion of 3-carbon

pyruvate to 2-carbon acetyl CoA, yielding a molecule of NADH and one CO2.
The acetyl CoA enters the citric acid or TCA cycle
Process
1. is transported into mitochondrial matrix across the inner mitochondrial
membrane
2. decarboxylated > forms a 2 C fragment (CH3COO-; acetyl group) + CO2
3. Acetyl group is transferred to coenzyme A to make acetyl CoA which carries
the 2 C fragment to Krebs cycle
4. Overall reaction: Pyruvate + HSCoA + NAD+ > Acetyl CoA + CO2 +
NADH + H+; catalyzed by a giant multienzyme complex (pyruvate
dehydrogenase)

TCA cycle is important

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accepts metabolites from or contributes them to other pathways entry point for
catabolic mechanism regardless of nature of starting material All of cell's
energy-providing macromolecules (polysaccharides, fats, proteins) break down to
TCA cycle metabolites
Acetyl CoA - breakdown product of fatty acids degraded 2 C's at a time
in matrix (fatty acids are also synthesized that way with acetyl CoA as

donor) & other catabolic pathways

Proteins break down to amino acids, which are catabolized & enter TCA
cycle at various sites; enter matrix via special transport systems in inner

mitochondrial membrane
reduced coenzymes in ATP formation (NADH & FADH2)
- are the primary product of pathway electrons they carry came from oxidized
-

Krebs substrates & used to make ATP

Glycolytic NADH
Enters mitochondrion (via malate-aspartate shuttle) reducing NAD+

NADH
Transfers electrons to FAD FADH2 (via glycerol phosphate shuttle)
Turning NADH & FADH2 ATP (Chemiosmosis_
Step 1
The electrons from NADH are transferred to the electron carrier

proteins & the protons are transferred across the membrane.

As electrons move from cytochrome to cytochrome, down the
electron transport chain, more protons are carried across the

membrane including the electrons from FADH2

Cytochrome c transfers electron to cytochrome c oxidase
complex. Protons are also transferred to the outside of he

membrane by the cytochrome c oxidase complex

The cytochrome oxidase complex then transferred electron from
cytochrome c to oxygen, the terminal electron acceptor that

formed water as the product.

The protons generate a proton motive force across the membrane
of the mitochondrion. Since membranes are impermeable to ions,
the protons that reenter the matrix pass through special proton
channel proteins called ATP synthase

Step 2

The energy derived from the movement of these protons is used

to synthesize ATP from ADP and P oxidative phosphorylation

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In

absence of

O2

fermentation
is carried
out in

cytoplasm

2.
Why
the TCA

is
cycle
considered
central pathway
energy metabolism of a cell?

to be the
in the
o

accepts metabolites from or contributes them to other pathways entry point for catabolic

mechanism regardless of nature of starting materials

All of cell's energy-providing macromolecules (polysaccharides, fats, proteins) break down to
TCA cycle metabolites
Acetyl CoA - breakdown product of fatty acids degraded 2 C's at a time in matrix
(fatty acids are also synthesized that way with acetyl CoA as donor) & other catabolic
pathways

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Proteins break down to amino acids, which are catabolized & enter TCA cycle at
various sites; enter matrix via special transport systems in inner mitochondrial
membrane

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3. Describe the steps by which the transport of electrons down the

respiratory chain leads to the formation of a proton gradient (see
Figure 5.17)

The energy released as electrons from NADH is pass down through the complexes of the
respiratory chain (I NADH dehydrogenase, III cytochrome c reductase, and IV cytochrome
c oxidase) to pump protons (H+) against their concentration gradient from the matrix of the

mitochondrion into the intermembrane space active transport

As their concentration increases there (which is the same as saying that the pH decreases), a
strong diffusion gradient is set up protons is passed through the ATP synthase complex
resulting the synthesis of ATP chemiosmosis and is an example of facilitated diffusion.

34 ATP are made from the products of 1 molecule of glucose.

The process is a stepwise movement of electrons from high energy to low energy that makes
the proton gradient

The proton gradient powers ATP production NOT the flow of electrons

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This electron transport chain only occurs when oxygen is available .

PROCESS
o Step 1
The electrons from NADH are transferred to the electron carrier proteins & the

protons are transferred across the membrane.

As electrons move from cytochrome to cytochrome, down the electron transport
chain, more protons are carried across the membrane including the electrons from

FADH2
Cytochrome c transfers electron to cytochrome c oxidase complex. Protons are
also transferred to the outside of he membrane by the cytochrome c oxidase

complex
The cytochrome oxidase complex then transferred electron from cytochrome c to

oxygen, the terminal electron acceptor that formed water as the product.
The protons generate a proton motive force across the membrane of the
mitochondrion (The number of hydrogen atoms (also called proton gradient) will
build up and flow back to the matrix simultaneously powering the production of
ATP) and dince membranes are impermeable to ions, the protons that reenter the
matrix pass through special proton channel proteins called ATP synthase

Step 2

The energy derived from the movement of these protons is used to synthesize
ATP from ADP and P oxidative phosphorylation

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4. What is the effect of Dinitrophenol on ATP formation by

mitochondria? Why is this case?

2,4-Dinitrophenol (2,4-DNP, or simply DNP), C6H4N2O5, is an inhibitor of efficient energy

(ATP) production in cells with mitochondria.

It uncouples oxidative phosphorylation by carrying protons across the mitochondrial

membrane, leading to a rapid consumption of energy without generation of ATP

How
o the energy source which drive the ATP synthesize is artificially removed by collapsing
the proton gradient ATP synthesis would stop but electron transport (oxidation of
o

NADH through formation of water from oxygen) would continue.

2,4-dinitrophenol can collapse the proton gradient and act as an uncoupler.
How proton gradient be collasped
a DNP molecule that approaches the inner mitochondrial membrane
from the outside becomes protonated (because the pH is lower there)
protonation increases the hydrophobicity of DNP, allowing it to diffuse
into the membrane and, by mass action, to pass through once inside,

the higher pH of the matric deprotonates the phenolic hydroxyl again.

Thus, DNP has the effect of transporting H+ back into the matrix,
bypassing the F0 proton channel and thereby preventing ATP synthesis.

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5. Describe the basic structure of the ATP synthase

The structure of ATP synthase consists of two rotary motors, labeled F1 and Fo, that are connected by a
flexible shaft.

Under normal operation, the Fo motor uses the energy stored in a transmembrane ion gradient to
drive the F1 motor in reverse so that ATP is synthesized from ADP and phosphate.

In bacteria, anerobic conditions wipe out the ion gradient whereupon the F1 part becomes a
motor, using the energy of ATP hydrolysis to turn the Fo part in reverse so that it functions as an
ion pump.

a.

Spherical F1 head (~90 diameter; very similar in mitochondria & bacteria, the structure
is highly conserved) - 360,000 daltons

Both bacterial & mitochondrial ATP synthases contain 5 different polypeptides (, , , ,)

with the following stoichiometry (33)

& subunits arranged alternately in F1 head like orange segments

Each F1 contains 3 catalytic sites for ATP synthesis (one on each subunit)

The subunit runs from outer tip of F1 head through central stalk & contacts the F0
basepiece

In the mitochondrial enzyme, all 5 F1 polypeptides are encoded by nuclear DNA, made in
the cytosol & imported into mitochondrion posttranslationally

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b. The F0 base is embedded in inner mitochondrial membrane; contains channel that conducts
protons from intermembrane space back to matrix presence of channel shown by
experiment

Break inner mitochondrial membrane into fragments forming membrane vesicles

(submitochondrial particles)

Intact submitochondrial particles containing ATP synthase embedded in vesicle membrane

can oxidize substrates, generate a proton gradient & synthesize ATP

If F1 spheres are removed from particles by urea treatment, vesicle membrane can no longer
maintain a proton gradient despite continuing substrate oxidation & electron transport

Protons translocated across membrane during electron transport simply cross back through
beheaded ATP synthase & energy is dissipated

6.
the
and
of

Describe
structure
function

peroxisomes and glyoxysome.

PERIXOSOME

organelles found in nearly all eukaryotic cells

single membrane microbodies found in photosynthetic cells of plants and liver and kidney cells of

vertebrates.
first discovered by J. Rhodin in 1954 and they were officially considered organelles in 1967 by

Christian de Duve.
contains enzymes like peroxides, oxidases and catalases
Structure
o are derived from the endoplasmic reticulum and replicate by fission

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o
o

surrounded by a lipid bilayer membrane which encloses the crystalloid core

the bilayer is enclosed with a plasma membrane which regulates what enters and exits the

peroxisome
There are at least 32 known peroxisomal proteins, called peroxins, which carry out
peroxisomal function inside the organelle.

Function
o to break down long and branched fatty acid chains, D-amino acids, and polyamines
using its enzymes transported to mitochondria where the majority of catabolism
happen (in both eukaryotic and prokaryotic cells; however in prokaryotes only happen in
o

peroxisome wihout the mitochondria)

site of catabolism of fats and fat-soluble vitamins, such as vitamin A and vitamin K, as

well as the production of bile acids

formation and degradation of hydrogen peroxide (H2O2) are able to break it down
into water (H2O), is harmless to the cell and oxygen (O2), can be used in the next

digestive
help in synthesizing plasmalogens and etherphospholipids that are necessary for

proper brain and lung function

aid certain enzymes with energy metabolism in many eukaryotic cells as well with

cholesterol synthesis in animals

involved in detoxification of poison in the cell, germinating seeds in the glyoxylate
cycle, photosynthesis in leaves, and oxidation of amines in various yeasts.

GLOXYSOME

also single membrane microbodies has similar structure as peroxisome but found only in plant

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cells
contains many enzymes like isocratic lyase, malate synthetase, glycolate oxidases, etc. for

glyoxylate cycle (i.e., metabolism of glycolic acid).

specialized peroxisomes found in plants (particularly in the fat storage tissues of germinating

seeds) and also in filamentous fungi.

Functions
o participate in photorespiration and nitrogen metabolism in root nodules.
o contain enzymes that initiate the breakdown of fatty acids and possess the enzymes to
produce intermediate products from the fats for the synthesis of sugars by
gluconeogenesis seedling uses these sugars synthesized from fats until it is mature
enough to produce them by photosynthesis and to generate new cell walls during growth

7. Explain the basics of ATP formation according to the binding

change mechanism Paul Boyer and Mitchel
The basics of ATP formation according to the binding change mechanism Paul Boyer, UCLA
(1979)
1. Energy released by proton movement changes active site binding activity for substrate &
product
o Energy released by proton movement does not directly phosphorylate ADP to ATP, but it
o

principally changes the active site binding affinity for the ATP product
In an aqueous environment where reactants & products are dissolved in medium, energy
is needed to drive the formation of the covalent bond linking ADP with inorganic

phosphate to form ATP

But when ADP & Pi bind to ATP synthase catalytic site, they condense to form a tightly

bound ATP molecule without the input of additional energy

Considerable energy (7.3 kcal/mole under standard conditions) is needed to form ATP

when reaction occurs in water solution (when ADP, PI & ATP are soluble).
2. Each active site goes successively through 3 distinct conformations that have different
substrate & product affinities the F1 complex has 3 catalytic sites, one on each of the 3
-subunits
o Properties of the 3 catalytic sites in a static enzyme (one not engaged in enzymatic
o

turnover), the different sites exhibited different chemical properties

Boyer proposed that at any one time, each of the 3 sites is present in different
conformations that have differing affinities for nucleotides: one in L, one in T, one in O
Loose (L) conformation - ADP & Pi are loosely bound
Tight (T) conformation - ADP + PI substrates or ATP product are tightly bound
Open (O) conformation it has a very low affinity for nucleotides allowing
release of tightly bound ATP; thus, it is considered empty

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3. ATP is produced by rotational catalysis - one part of ATP synthase rotates relative to
another part
o Boyer proposed that the & subunits, which form a hexagonal ring of subunits within
o

the F1 head, rotate relative to the central stalk ( rotational catalysis)

Rotation is driven by proton movement through membrane into matrix via the channel

inF0 base
Thus, electrical energy stored in proton gradient is transduced into the mechanical energy
of a rotating stalk, which is transduced into chemical energy stored in ATP