Biochemistry Section E (1 and 2)

What are the main sources of Acetyl CoA?

Proteins, Glucose and Fatty Acids.

Glucose and Fatty acids are larger contributors via the break down of each of them (catabolism)

What do all three sources give off when producing Acetyl CoA?

high energy electrons that can be utilized to produce energy

What feeds into the Krebs Cycle? What is the most basic component produced?

Acetyl CoA; CO2

What is step one in cellular respiration?

Oxidation (breakdown) of fuels (food) into Acetyl CoA which generates ATP and reducing equivalents (NADH/FADH2)

How does oxidation of fuels generate the ATP?

Electrons move from high-energy component to lower energy component. As the electrons move, the energy is incorporated into the generation of ATP/reducing agents.

What is step two in cellular respiration?

Oxidation (breakdown) of Acetyl CoA to CO2 in the citric acid cycle

What does the citric acid cycle release while oxidizing Acetyl CoA?

As Acetyl CoA is oxidized throughout many steps, it releases many high-energy electrons that can aid in the generation of NADH/FADH2/GTP which can be fed into the electron transport chain.

What is step three of cellular respiration?

The reducing agents produced will pass the electrons into the electron transport chain that generates the ATP required to fuel cellular systems.

What provides the most amount of ATP?

Krebs cycle to the electron transport chain. Glycolysis alone does not generate enough.

What is the end product of glycolysis? Where does it go for further catabolism?

pyruvate; enters the mitochondria via pyruvate carrier

What two pathways can pyruvate take once in the mitochondria?

Pyruvate dehydrogenase and pyruvate carboxylase

What is the pyruvate dehydrogenase complex?

generates Acetyl CoA that feeds into the Krebs cycle, main regulatory pathway that determines if pyruvate will be oxidized or not.

How does pyruvate dehydrogenase complex act as a regulator?

Exhibits feedback inhibition. Once we have sufficient energy in our system, the products it generates (Acetyl CoA and NADH) will shut it down because they are in excess.

What is the pyruvate carboxylase pathway?

generates oxaloacetate that feeds into the Krebs cycle, generated when we have an excess of Acetyl CoA. Another method of regulation.

What types of modulators are pyruvate dehydrogenase and carboxylase?

Allosteric- feedback inhibition

How does pyruvate turn into Acetyl CoA once in mitochondria?

Uses the pyruvate dehydrogenase pathway. Interacts with CoA-SH that will remove the carboxyl group on pyruvate and add an S-CoA group, turning it into Acetyl CoA and CO2.

What is the reaction called when pyruvate turns into Acetyl CoA via pyruvate dehydrogenase?

Oxidative Decarboxylation, irreversible

How does pyruvate become trapped in the mitochondria?

When turned into acetyl-CoA, there are no transporters that allow it to be transported out. Allows for more catabolism to be performed.

How does the trapping of Acetyl-CoA modulate pyruvate dehydrogenases activity?

You can not keep trapping an excess amount of Acetyl-CoA in the mitochondria. When too much is formed, it shuts down dehydrogenase complex.

What are some factors that turn PDH off?

ample fatty acids and Acetyl-CoA available

ample ATP/ADP and NADH/NAD

What are some factors that turn PDH on?

energy demands are high and cell requires greater flux of Acetyl-CoA into Krebs cycle.

What is another type of modulator that pyruvate dehydrogenase can be?

Covalent modification via phosphorylation

What is PDH kinase?

the enzyme will add a phosphate to PDH and inhibits its activity

How is the PDH kinase activated? Deactivated?

Allosterically by products of the complex (ATP, NADH, Acetyl-CoA); by substrates of the complex (ADP, NAD+, Pyruvate)

What is PDH phosphatase?

Takes the phosphate off of PDH and reverses the inhibtion

How is Acetyl-CoA made by pyruvate dehydrogenase?

From Coenzyme A (CoA-SH). The reactive thiol group will bind to pyruvate to make Acetyl-CoA via a thioester bond.

How does the addition of the thioester bond trap Acetyl-CoA in the mitochondria?

When turned into Acetyl-CoA, there are no transporters that allow it to be transported out.

What is oxaloacetate required for in the Krebs Cycle?

For the oxidation of Acetyl-CoA being generated by pyruvate dehydrogenase complex. It will digest the excess Acetyl-CoA

What is the overall role of the Krebs cycle regarding oxaloacetate?

During the Krebs cycle, oxaloacetate is being regenerated while acetyl-CoA is being broken down into substrates.

What is the first critical step in the citric cycle?

Converting acetyl-CoA to citrate via the reaction between oxaloacetate and acetyl-CoA. Catalyzed by citrate synthase.

What is another critical step in the citric acid cycle?

The conversion of isocitrate to alpha-ketoglutarate. Catalyzed by isocitrate dehydrogenase.

What is the final critical step in the citric acid cycle?

Conversion of alpha-ketoglutarate to succinyl CoA. Catalyzed by alpha-ketoglutarate dehydrogenase.

What is dehydrogenase?

an enzyme that catalyzes by removing hydrogens from compounds (collecting the electrons)

What is being produced during the critical oxidation steps of the citric acid cycle?

Energy and reducing equivalents such as NADH and FADH2

How is the production of NADH/FADH2 related to thermodynamics?

It is a thermodynamically favorable reaction, release of energy from high to low is transformed to NADH/FADH2

What three steps in the citric acid cycle are irreversible?

citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase.

What is inhibitory of citrate synthase? Stimulatory?

NADH, succinyl-CoA, citrate, ATP; ADP

What is inhibitory to isocitrate dehydrogenase? Stimulatory?

ATP; CA2+ and ADP

What is inhibitory to alpha-ketoglutarate dehydrogenase? Stimulatory?

succinyl-CoA and NADH; Ca2+

What are the products of the energy released from the citric acid cycle?

3 NADH, 1 FADH2, 1 GTP (ATP)

How many ATP molecules are generated during Krebs cycle?

1, not sufficient for cellular function

How is more energy produced?

NADH and FADH2 feed into the ETC.

NADH produces 2.5 ATP and FADH2 produces 1.5 ATP

What are anaplerotic reactions?

chemical reactions that replenish intermediates in citric acid cycle

What are the two intermediates of the citric acid cycle that are regenerated by anaplerotic reactions?

Oxaloacetate and malate

What is an amphibolic pathway?

one that serves as both catabolic and anabolic

What is an example of an amphibolic pathway? Why?

Krebs cycle; generates intermediates but also consumes Acetyl-CoA

What are the only two anaplerotic steps in the Krebs cycle?

Pyruvate to oxaloacetate and pyruvate to malate

Is pyruvate carboxylase anabolic or catabolic? What is it used in?

Anabolic; gluconeogenesis

What is the role of thiamine pyrophosphate?

It is a cofactor for pyruvate dehydrogenase. It is required to cleave C-C bonds that lead to the formation of acetyl-CoA from pyruvate. Decarboxylation

What is the role of biotin?

It is a cofactor for pyruvate carboxylase. It is required for the formation of a C-C bond that leads to the formation of oxaloacetate from pyruvate. Carboxylation

Why is oxygen so important?

Critical driver of energy production

How many membranes does the mitochondria have?

2

Describe the outer membrane of the mitochondria

The outer membrane is highly permeable due to many large pores that allow the passage of ions and small proteins.

Describe the inner membrane of the mitochondria

Impermeable to most small molecule and ions, transport requires transporters.

What is found in the inner membrane of the mitochondria?

the electron transport chain complexes, ATP synthase complex, the pyruvate transporter, the citrate transporter, and other transporters that permit a limited number of molecules to pass through the inner mitochondrial membrane.

What is the matrix of the mitochondria?

Found inside the inner membrane.

What is in the matrix of mitochondria?

PDH complex, enzymes for Krebs cycle, enzymes of fatty acid oxidation, and enzymes for amino acid oxidation.

Remember, pyruvate is move into the mitochondria to be oxidized to acetyl-CoA

What does it mean to be reduced? Oxidized?

Adding a hydrogen to the molecule; Removing a hydrogen from the molecule

What is the Respiratory ETC?

series of electrons carriers

How does dehydrogenase aid in ETC?

Collects electrons from catabolic pathways and funnel them into universal electron acceptors: NAD and FAD

What is a reducing agent? Oxidizing agent?

Enzyme that catalyzes oxidation; enzyme that catalyzes reduction

What are the three types of electron transfers?

Direct transfer of electrons, transfer as a hydrogen atom, and transfer as a hydride ion

What is a reducing agent?

Electrons transferred to these molecules that generates reducing equivalents. NADH and FADH2

How many electrons does NADH carry to ETC?

2

What are the 5 types of electron-carrying molecules (reducing agents)?

NAD, FAD, ubiquinone, cytochromes, and iron-sulfur proteins

What electron-carrying molecules are apart of the ETC?

ubiquinone, cytochromes, and iron-sulfur proteins

How many complexes are in the ETC?

4 complexes

Where does NADH enter the electron transport chain?

Enters at complex one and transfers electrons to the chain. Becomes NAD again. Electrons then move to CoQH2 to 3 then to 4.

Where does FADH2 enter the electron transport chain?

Enters at complex two and transfers electrons to the chain. Electrons then move to 3 to 4.

Why does FADH2 produce less ATP than NADH?

Enters the ETC at the second complex rather than the first.

Why is oxygen important in the electron transport chain?

It is the final acceptor of electrons at the end of the transport chain. It is then converted to water. Without acceptor, the chain will shut down.

How is the order of molecules going through the electron transfer chain determined?

Compounds with large negative standard redox potentials are at the start because they have a strong tendency to release electrons. The final acceptor has positive potential and will accept electrons.

What other pathway products donate to ETC in complex 2?

succinate and electrons from fatty acid oxidation

How does rotenone (rat poison) affect the ETC?

Stops electron transport at complex one

How does antimycin A affect the ETC?

Stops electron transport at complex three

How does cyanide or carbon monoxide affect the ETC?

Stops electron transport to final acceptor (oxygen)

Describe reactive oxygen species.

The oxygen becomes a super radical ion (superoxide) that will damage ETC and proteins because it is so highly negative.

How do we control reactive oxygen species?

Superoxide dismutase will catalyze conversion of superoxide to hydrogen peroxide. Hydrogen peroxide is then catalyzed to water by glutathione peroxidase.

What can lead to ROS production?

Can occur during hypoxia, occurs due to a mismatch between electrons donors and acceptors. Oxidative stress

What are the two defenses against oxidative stress or superoxide ions?

1. glutathione peroxidase

2. regulation of pyruvate dehydrogenase, phosphorylated (inactive) in hypoxic conditions

What do low ROS levels indicate?

Insufficient oxygen levels

How is ATP produced from ETC?

The energy released via the highly exergonic reaction is used to synthesize ATP

Describe how a voltage gradient is generated from ETC. Why is it important?

As you are transferring electrons in ETC, you are pushing protons into the intermembrane space. This generates a voltage gradient since the inner mitochondria membrane is negative and the space between membranes is now positive; This fuels ATP synthesis.

Explain why the voltage gradient synthesized ATP

Protons will move down the gradient into the cell through an ATP synthase thus generating ATP.

What are the three components of the ATP synthase?

Adenine nucleotide translocase, phosphate translocase, and ATP synthasome.

What does the adenine nucleotide translocase do?

It is an antiporter that moves ADP into the matrix to create more ATP and moves ATP out of the cell.

What does the phosphate translocase do?

It is a symport that moves hydrogen and phosphate into the matrix.

What does the ATP synthasome do?

A complex of ATP synthase and both translocases. It will move protons into the matrix and synthesize ATP.

How are NADH moved from cytosol into matrix to donate to the ETC?

Two shuttles:

malate-aspartate shuttle (complex 1) and glycerol 3-phosphate shuttle (complex 3)

What is oxidative phosphorylation?

Using oxidation processes through transport of electron in ETC. Use the energy produced to add a phosphate to ADP to create ATP.

Is succinate or fumarate reduced?

Succinate

What does brown adipose tissue do?

Functions to generate heat through fuel oxidation

How does the uncoupling protein 1 make the ETC produce heat instead of energy?

It will break the chain by adding the UCP. Electrons will now flow through the UCP and this prevents voltage gradient from forming. The voltage gradient creates ATP, so now instead of energy, we have heat.

How do uncoupling proteins affect oxygen consumption and ATP synthesis?

They dissipate the proton gradient and will not allow ATP to form. The electron transport chain is activated to try and increase ATP, this increases oxygen consumption.

How do electron transport inhibitors affect oxygen consumption and ATP synthesis?

They completely inhibit the electron transport chain and do not allow a proton gradient to form. There is no increase in oxygen because the ETC is not activated.