9 - Pyruvate Fate, Citric Acid Cycle, and Electron Transport Chain

These flashcards cover the fates of pyruvate (anaerobic vs aerobic), the role of Acetyl-CoA, the steps and regulation of the Citric Acid Cycle, and the mechanisms of the Electron Transport Chain and ATP synthesis as discussed in Lecture 9.

In the 'Big Picture' overview, where does glycolysis occur in the cell?

Cytosol

In the mitochondrion, what is the net ATP yield specifically from the Citric Acid (Krebs) cycle per glucose molecule?

$2$ ATP

What is the approximate range of ATP molecules generated by the Electron Transport Chain per glucose molecule?

$32$-$34$ ATP

What factor primarily determines the metabolic fate of pyruvate?

The availability of oxygen

Why must NADH be re-oxidized during cellular metabolism?

Cells have a limited supply of $NAD^+$ and it must be replenished to allow for continued glycolysis.

In the presence of oxygen, where is NADH passed for re-oxidation?

Into the mitochondria via the Electron transport chain

Under anaerobic conditions, how is $NAD^+$ replenished in the cytoplasm?

Through the reduction of pyruvate in an extension of the glycolytic pathway.

What is the name of the anaerobic process by which humans convert pyruvate to regenerate $NAD^+$?

Homolactic fermentation

Under what muscle conditions does homolactic fermentation typically occur?

During vigorous activity when ATP demand is high and oxygen is depleted.

Which enzyme catalyzes the oxidation of NADH by pyruvate to yield $NAD^+$ and lactate?

Lactate dehydrogenase (LDH)

How is lactate exported from a muscle cell into the blood?

Via Lactate-$H^+$ transporters, also known as monocarboxylate transporters.

To which organ is lactate transported to be reconverted into glucose?

The liver

What is the name of the cycle where lactate is converted to glucose in the liver and returned to the muscle?

Cori/Lactic acid cycle

What are the substrates, enzyme, and products for homolactic fermentation?

Substrate: Pyruvate; Enzyme: Lactate dehydrogenase (LDH); Product: Lactate and $NAD^+$.

What two products do yeast produce from pyruvate during alcoholic fermentation?

Ethanol and $CO_2$

What is the first reaction in alcoholic fermentation in yeast, and which coenzyme does it require?

Decarboxylation of pyruvate to form acetaldehyde and $CO_{2}$, using thiamine pyrophosphate (TPP).

What is the second reaction in alcoholic fermentation in yeast?

Reduction of acetaldehyde to ethanol and $NAD^+$.

Where does the aerobic oxidation of pyruvate and generation of acetyl-CoA occur?

Mitochondria

Which multi-enzyme complex is responsible for the aerobic oxidation of pyruvate?

Pyruvate dehydrogenase complex

During the conversion of pyruvate to acetyl-CoA, what happens to the released electron?

It is released to $NAD^+$, which is reduced to NADH.

What is the name of the protein family identified as essential for pyruvate uptake into the mitochondria?

Mitochondrial Pyruvate Carrier (MPC)

Where specifically are MPC proteins localized within the mitochondria?

The inner mitochondrial membrane (IMM)

Which two proteins form the heterocomplex of the mitochondrial pyruvate carrier in yeast and mammals?

Mpc1 and Mpc2

What growth defect do yeast cells lacking MPC1 exhibit?

A severe defect in mitochondrial pyruvate uptake and growth in synthetic dextrose (SD) medium.

Which specific amino acids can restore growth of MPC mutant yeast strains in synthetic dextrose?

Valine or leucine

What is the approximate size of the MPC complex discovered using Blue native-polyacrylamide gel electrophoresis?

$150$ kilodalton ($kDa$).

What human genetic conditions are associated with causal loci mapped to MPC1?

Lactic acidosis and hyperpyruvatemia

What inhibitor is known to act specifically and potently on mitochondrial pyruvate carrier activity?

$\text{UK-5099}$ (a-cyanocinnamate analogs)

What are the three components of the Pyruvate Dehydrogenase (PDH) complex?

Pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3).

How many NADH molecules are formed per glucose molecule during the PDH reaction?

$2$ NADH

What specific task is performed by the E1 component of the PDH complex?

It makes TPP + 2 carbons and releases $CO_2$.

What product is generated by the E2 component of the PDH complex?

Acetyl-CoA

What product is generated by the E3 component of the PDH complex?

NADH

Which ion is required for the attachment of the 2-carbon molecule to TPP in the PDH complex?

$$Mg^{2+}$$

What are the summary substrates and products of the Pyruvate conversion to Acetyl-CoA?

Substrate: Pyruvate; Product: Acetyl-CoA.

How is the E1 component of the PDH complex inactivated?

Via phosphorylation by PDH kinase.

What factors activate PDH kinase, leading to PDH inactivation?

High levels of ATP, NADH, or acetyl-CoA.

Which enzyme reactivates PDH by removing the phosphate group?

PDH phosphatase

Which product inhibits the E2 component of PDH?

Acetyl-CoA

Which product inhibits the E3 component of PDH?

NADH

What is the definition of metabolism as provided in the lecture?

The process through which we acquire and utilize free energy.

What is the primary reason for performing metabolism?

To make ATP

What is Coenzyme A (CoA) with an acetyl group attached called?

Acetyl-CoA

What percentage of all enzymes are estimated to use Coenzyme A (CoA)?

$$4\%$$

What type of high-energy linkage connects the acetyl group to Coenzyme A?

Thioester linkage

What pathways can produce Acetyl-CoA?

Fatty acid metabolism, amino acid metabolism, and glycolysis.

How many carbons enter the Citric Acid Cycle in the form of acetyl-CoA?

$2$ carbons

What is the initial 6-carbon molecule formed in the Citric Acid Cycle?

Citrate

Per cycle, what are the specific products generated by the Citric Acid Cycle?

$2$ $CO_2$, $3$ NADH, $1$ ATP, and $1$ $FADH_2$.

Per mole of glucose, how many NADH and $FADH_2$ are generated by the Citric Acid Cycle?

$6$ NADH and $2$ $FADH_2$.

What can be the sources for Acetyl-CoA used in the Citric Acid Cycle?

Carbohydrates, fats, and proteins.

Why is the Citric Acid Cycle described as 'amphibolic'?

It is both catabolic (breakdown) and anabolic (providing intermediates for de novo synthesis).

Where in the mitochondrion does the Citric Acid Cycle occur?

Mitochondrial matrix

What are the substrates and enzyme for 'Reaction 1' of the Citric Acid Cycle?

Substrates: Oxaloacetate ($4$ carbons) + Acetyl-CoA ($2$ carbons); Enzyme: Citrate synthase.

What reaction ensures the Citric Acid Cycle proceeds even at low concentrations of oxaloacetate?

The highly exergonic hydrolysis of the thioester bond in the citrate intermediate.

Which enzyme catalyzes Citrate isomerization to Isocitrate?

Aconitase

Which crucial cofactor is used by the enzyme aconitase?

An iron-sulfur cluster

What are the substrate, enzyme, and product for 'Reaction 3' of the Citric Acid Cycle?

Substrate: Isocitrate; Enzyme: Isocitrate dehydrogenase; Product: Alpha-ketoglutarate (+NADH).

What is the rate-limiting step of the Citric Acid Cycle?

The reaction catalyzed by isocitrate dehydrogenase.

What is the standard free energy change ($\Delta G^{0'}$) for the isocitrate dehydrogenase reaction?

$-11.6$ $kJ/mol$

Which enzyme catalyzes the conversion of alpha-ketoglutarate to succinyl-CoA?

Alpha-ketoglutarate dehydrogenase complex

Which reaction produces the only ATP (or GTP) directly within the Citric Acid Cycle?

Reaction 5: Succinyl-CoA conversion to succinate by succinyl-CoA synthetase.

Which enzyme converts between ATP and GTP within the cell?

Nucleosidediphosphate kinase

Which enzyme catalyzes the formation of fumarate from succinate?

Succinate dehydrogenase

Where is succinate dehydrogenase located and how does it deliver electrons?

It is membrane-bound and delivers electrons directly into the ETC via coenzyme Q.

What type of double bond is produced in the formation of fumarate?

Trans double bond

Which hydrolytic enzyme converts Fumarate to L-malate?

Fumarase

Which enzyme catalyzes the final reaction (Reaction 8) of the Citric Acid Cycle to reform oxaloacetate?

Malate dehydrogenase

What is the $\Delta G^{0'}$ of the malate dehydrogenase reaction?

$+29.7$ $kJ/mol$

Why does the endergonic formation of oxaloacetate proceed in the cell?

Because the subsequent reaction (citrate synthase) is highly exergonic, keeping oxaloacetate levels very low.

What is the net yield of ATP and electron transporters for one molecule of glucose through aerobic glycolysis and the Citric Acid Cycle?

$4$ ATP, $10$ NADH, and $2$ $FADH_2$.

What two points primarily control entry into the Citric Acid Cycle?

Pyruvate dehydrogenase (PDH) and citrate synthase.

What are the two key irreversible reactions regulated within the Citric Acid Cycle?

Isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase.

Cycle flux is primarily controlled by which three factors?

Allosteric activation by ADP, the $NAD^+/NADH$ ratio, and inhibition by acetyl-CoA or succinyl-CoA.

What is the final step in aerobic respiration and what are its two processes?

Electron transport chain (transporting electrons) and oxidative phosphorylation (ADP to ATP).

How many protein complexes make up the Electron Transport Chain (ETC)?

$4$ protein complexes

Where do the protein complexes of the ETC reside?

Inner mitochondrial membrane (IMM)

What is the function of the electrochemical gradient created by the ETC?

It creates a proton motive force that leads to the creation of ATP via ATP synthase.

Which mitochondrial compartment is freely permeable to small molecules ($< 5\,kDa$)?

Outer mitochondrial membrane (OMM)

Name four types of coenzymes/carriers utilized by ETC proteins.

Flavoproteins ($FMN$/$FAD$), iron-sulfur proteins ($FeS$), Coenzyme Q, and Cytochromes ($heme$).

What is 'standard reduction potential'?

A measure of the ability to accept or donate electrons.

Which molecule has the highest standard reduction potential and is the final electron acceptor in the ETC?

Oxygen ($O_2$)

How does the ETC flow electrons in terms of reduction potential?

From low reduction potential carriers to high reduction potential carriers.

What is the name of ETC Complex I?

NADH–coenzyme Q reductase

How many iron-sulfur clusters are contained in Complex I?

$8$ iron-sulfur clusters

How many protons are pumped by Complex I for every two electrons donated by NADH?

$4$ protons

What is the name of ETC Complex II?

Succinate–Coenzyme Q Reductase

Why is Complex II unique compared to the other complexes regarding its pumping activity?

Complex II does not pump $H^+$ protons.

What is the name of ETC Complex III?

Coenzyme Q - cytochrome c oxidoreductase

How many cytochrome c molecules are reduced in the process of Complex III?

$2$ cytochrome c molecules

How many protons are pumped into the intermembrane space by Complex III?

$4$ protons

What is the name of ETC Complex IV?

Cytochrome c oxidase

For every two electrons transferred, how many protons does Complex IV pump into the intermembrane space?

$2$ protons

In the net reaction for four electrons in Complex IV, how many protons are consumed to form water?

$4$ protons

What two changes define the electrochemical gradient across the inner mitochondrial membrane?

Drop in pH and an increase in voltage difference.

What provides the free energy for ATP synthesis through complex V?

The dissipation of the electrochemical gradient.

What is another name for Complex V?

ATP synthase (or the $F_0 F_1$ complex)

Which subunit of ATP synthase is embedded in the membrane and acts as an ion channel?

The $F_0$ subunit

Which subunit of ATP synthase protrudes into the matrix and is responsible for ADP phosphorylation?

The $F_1$ subunit

What are the components of the '$F_1$ knob' in ATP synthase?

Three $\alpha\beta$ dimers arranged around a central stalk (rotor) consisting of $\gamma$, $\delta$, and $\epsilon$ subunits.