ASSIGNMENT ON THE CITRIC ACID CYCLE, RESPIRATORY CHAIN AND OXIDATIVE PHOSPHORYLATION

1. Explain why the Citric Acid Cycle is an amphibolic pathway. How is it related to carbohydrate, fat and protein
metabolism?

The term amphibolic is used to describe biochemical pathways that would involve catabolism and anabolism.
Catabolism being the degradative part of metabolism in which large molecules are converted to simple molecules.
This is the breaking apart of molecules to smaller one in order for energy to be released. Example of such reaction
are digestion and cellular respiration where in sugars and fats are broken down for energy, breakdown from
protein to amino acids, triglycerides to fatty acids and disaccharide into monosaccharides. This involves two types
of reaction hydrolysis reaction and oxidation reactions (removal of hydrogen and electrons from organic
molecules. Anabolism, on the other hand, involves the biosynthesis phase of metabolism in that smaller and
simpler molecules are converted to larger more complex molecules. This involves the formation of carbohydrates,
proteins, lipids and nucleic acids. Reactions involved are dehydration synthesis reactions and reduction reactions.
This process allows molecules to gain energy. The term amphibolic was used to emphasize the dual nature of the
pathway. This pathways serves as the central metabolic pathway. All reaction associated with biochemical
synthesis converge into common pathways such as glycolysis, Kreb’s cycles and electron transport chain exist as an
amphibolic pathway.

Carbohydrates are organic molecules composed of C, H and O atoms. This family of carbohydrates include simple
and complex sugars. Cells utilized different steps in breaking down absorbed glucose to carbon dioxide and water
through a variety of enzymatic reaction. Catabolism of glucose involves two metabolic pathways – glycolysis and
tricarboxylic acid / citric acid cycle/ Kreb’s cycle. Free circulating glucose is taken up in the body in response to
insulin and a form of reaction called glycolysis which simply involves the transfer of energy from glucose to ADP to
form ATP. In this process the last step would be the production of pyruvates. In the presence of adequate oxygen,
pyruvate will further proceed to the Kreb’s cycle where in additional energy will be produced.

Protein metabolism involves the breakdown of protein molecule to amino acid and other simpler derivative
compounds, for transport mechanism through the cell membrane and ultimately the polymerization into new
proteins through the use of RNA and ribosome. The primary purpose of protein metabolism is so that organism can
convert proteins into a form of energy that the body will be able to utilize. In order to convert to energy, protein
are typically deaminated so that they can be process into the Citric acid cycle. In here original proteins will be
converted to more usable form of energy for the cell. Specific amino acids can enter in the different steps of the
Kreb’s cycle.

Fatty acid metabolism involve the catabolic process that generate energy and an anabolic process that creates vital
biological molecules such as triglycerides, phospholipids, hormones etc. Fatty acids yields the most ATP on an
energy mass per gram basis when completely oxidized to CO2 and water by beta oxidation and citric acid cycles.
Beta oxidation is a repeated process among fatty acids until it has been reduced to Acetyl CoA. The acetyl CoA
produced would be the one to enter the citric acid cycle in the mitochondrion by combining with oxaloacetate to
form citrate.

2. What are anaplerotic reactions and what is their importance particularly in the CAC? Cite an example.

Anaplerotic means the replenishment of reaction. Various intermediates of the citric acid cycle are also used for
the synthesis of other substances. Anaploresis in turn is defined as any reaction that can restore the concentration
of crucial intermediates that have been depleted. Several biosynthetic pathways uses intermediate in the TCA as
starting materials. Pyruvate carboxylase is a major anaplerotic enzyme. The citric acid cycle operates when
oxaloacetate is available. In animal tissue (liver and kidney) oxaloacetate is formed by the carboxylation of
pyruvate by pyruvate carboxylase in the mitochondria. Pyruvate carboxylase catalyzes the addition of CO2 to
pyruvate to form oxaloacetate. This enzyme is activated by acetyl CoA ad is inhibited by high concentration of
many acyl CoA derivatives. As the concentration of oxaloacetate is depleted through the efflux of TCA cycle
intermediates, the rate of citrate synthase reaction decreases and acetyl CoA concentration rises. The acetyl CoA
then activates pyruvate carboxylase to synthesize more oxaloacetate.

3. Explain the chemiosmotic theory in the production of ATP. Describe how the electron transport chain generates
a proton concentration gradient.

The chemiosmotic theory was proposed by Peter Mitchell which states that a proton-motive force is responsible
for driving the synthesis of ATP. Protons are pumped across the inner membrane of the mitochondria as electrons
went through the electron transport chain. This result in a proton gradient with a lower pH in the intermembrane
space and an elevated pH in the matrix of the mitochondria. In this model an intact inner mitochondrial membrane
impermeable to protons is a requirement. The proton gradient and membrane potential are the proton-motive
force that is used in order to drive ATP synthesis. In effect, the pH gradient acts as the “battery” that stores energy.
This theory has been the most widely accepted mechanism of coupling of electron transport and ATP synthesis.

Proof of this theory includes the electron generates a proton gradient, the pH measure on the outside of the
mitochondria is lower than that of the inside. Only a proton gradient is needed to synthesize ATP. Electron
transport is not required as long as there is an existing mechanism for generating a pH gradient

This theory suggest that most ATP synthesis in respiring cells comes from the electrochemical gradient across the
inner membrane of the mitochondria by using the energy of NADH and FADH2 from the breakdown of energy rich
molecules such as glucose. The carriers pass electrons to the electron transport chain in the inner mitochondrial
membrane which in turn pass them to other proteins in the ETC. The energy available in electrons is used to pump
protons from the matrix across the stroma.

As electrons move energetically downhill. The complexes capture the released energy and used it to pump H ion
from the matrix to the intermembrane space. The pumping of H forms an electrochemical gradient across the
inner mitochondria membrane. This gradient is sometimes call the proton-motive force. This movement of ions are
dependent on the combination of two factors p diffusion and electrostatic force.

4. Describe the structure of ATP Synthase/Complex V and the role of proton motive force in its function.

Human mitochondrial ATP synthase or complex V consist of two functional subunits, namely F1 and F0. F1 is
comprised of 5 different subunits (three α, three β, and one γ, δ and ε) and is situated in the mitochondrial matrix.
F0 contains subunits c, a, b, d, F 6, OSCP (oligomycin sensitivity-conferring protein) and the accessory subunits e,f,
g and A6L. F1 subunits γ, δ and ε comprises the central stalk of complex V. Subunits b, d, F6 and OSCP forms the
peripheral stalk. Protons pass from the intermembrane space to the matrix through the F0 which transfers the
energy created by the proton electrochemical gradient to F1, where in ADP is phosphorylated to ATP.
The function of the ATP synthase is to synthesize ATP from ADP and inorganic phosphate I nthe F1 sectors. This is
possible due to the energy derived from the gradient of protons which crosses the inner mitochondrial membrane
from the intermembrane space into the matrix through the F0 portion. The proton gradient is the one who
establishes a proton motive force which has 2 components – pH differential and electrical membrane potential.
The released energy causes the rotation of two rotary motors the ring of c subunits in the F0 and the subunits γ, δ
and ε in F1. Protons pass F0 via subunit a to the C ring. Rotation of the gamma subunit within the F1 provides
energy for ATP synthesis. The binding change mechanism describe ATP synthesis and ATP hydrolysis occurs at the
catalytic sites which are found in the three beta subunits. Each site switches cooperatively through conformation in
which ADP and Pi bind.

5. What are uncouplers? Explain their mechanism of action and give some examples. How are they beneficial in
specific circumstances?

Uncouplers or uncoupling agents are molecules that causes a disruption in oxidative phosphorylation by
dissociating the reaction of ATP synthesis from the electron transport chain. This results is that the cell or
mitochondria expend energy to generate a proton motive force but the proton motive force is dissipated before
the ATP synthase can recapture the energy and utilize to make ATP. This compounds causes an increase in
permeability of the inner mitochondrial membrane resulting to a failure in establishing an electrochemical
gradient. They act by transporting hydrogen to inside mitochondria without passing through the F0 and F1.

Once example would be Dinitrophenol which is has been known as a dieting aid but was associated with several
side-effects and event deaths. DNP acts as a protonphore which allows the leaking of protons across the inner
mitochondrial membrane which bypasses the ATP synthase which makes the energy production of ATP less
efficient. Ad an effect the energy that is produces from cellular respiration is wasted in the form of heat. DNP
causes a release of calcium from mitochondrial stores and prevent calcium reuptake which leads to muscles
contraction and hyperthermia.

One beneficial use of uncouplers is that it can generate heat though as a wasteful byproduct. This usually occurs in
many hibernating animals and in newborns and mammals adapted to cold. This occurs in specialized brown
adipose tissue. The uncoupling protein called thermogenin can accomplish this uncoupling and allow heat
generation.

6. Explain how respiratory control ensures a constant supply of ATP to the organism

Under most physiologic conditions, electron transports is tightly couple to phosphorylation. Electrons to not
usually flow through the ETC to oxygen unless ADP I simultaneously phosphorylated to ATP. Oxidative
phosphorylation would require a supply of NADH, oxygen, ADP and Pi. Though the most important factor that
determines the rate of oxidative phosphorylation is the ADP levels. The consumption rate of oxygen by the
mitochondria is increased markedly when ADP is added and then would go back to its initial value when the added
ADP has been converted to ATP. The regulation of oxidative phosphorylation by the levels of ADP is called the
respiratory control or acceptor control. ADP levels also affects the rate of the citric acid cycle due to the need for
NAD+ and FAD. ADP levels increases when ATP is consumed and so oxidative phosphorylation is couple to the ATP
utilization. Electron would not flow from fuel molecules to oxygen unless there is a need for ATP to be synthesized

https://courses.lumenlearning.com/boundless-microbiology/chapter/the-citric-acid-krebs-cycle/

https://bajan.files.wordpress.com/2010/05/amphibolic-nature-of-krebs-cycle.pdf

https://opentextbc.ca/anatomyandphysiology/chapter/24-2-carbohydrate-metabolism/

https://open.oregonstate.education/animalnutrition/chapter/chapter-5/
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metabolic-pathways/

https://www.nature.com/scitable/topicpage/nutrient-utilization-in-humans-metabolism-pathways-14234029/

https://www.letstalkacademy.com/publication/read/regulation-of-the-citric-acid-cycle-anaplerotic-reactions

https://www.khanacademy.org/science/ap-biology/cellular-energetics/cellular-respiration-ap/a/oxidative-
phosphorylation-etc#:~:text=The%20proton%20gradient%20generated%20by,in%20the%20inner
%20mitochondrial%20membrane

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278611/#:~:text=ATP%20synthase%20consists%20of
%20two,subunits%20%CE%B3%2C%20%CE%B4%20and%20%CE%B5.

https://www.slideshare.net/nomank992/uncouplers-of-oxidative-phosphorylation

http://fig.cox.miami.edu/~cmallery/255/255etc/chemiosm.htm

http://www.biology.arizona.edu/biochemistry/problem_sets/metabolism/04c.html#:~:text=Dinitrophenol
%20(DNP)%20is%20an%20uncoupler,to%20help%20patients%20lose%20weight.

https://www.ncbi.nlm.nih.gov/books/NBK22448/