Exam III: Chapter 17 (Citric Acid Cycle)

What enzyme is responsible for the hydration of Fumarate? Where does Fumarate come from? Please draw the mechanism!

Fumarate is the product of succinate oxidation by Succinate Dehydrogenase. Fumarase is the enzyme responsible for the hydration of Fumarate, which creates L-Malate. An OH- group binds to the double bond of Fumarate, creating L-Malate.

What makes this coordinated catalysis possible?

1.The structural integration of three kinds of enzymes 2. The long, flexible, lipoamide arm 3. Proximity of one enzyme to another All of these make coordinated catalysis possible, and increase reaction rates while minimizing interferences.

What makes FADH2 different from NADH?

FADH2 can accept two electrons, and is usually bound to the enzyme (Succinate Dehydrogenase), unlike NADH that which dissociates from the reaction.

How many CO2 are released from the two rounds of the Citric Acid Cycle?

Four CO2's

RECALL: Where does Glycolysis occur, again?

Glycolysis occurs in the cytoplasm.

When ATP is high what does PDK and PDP do to Pyruvate Dehydrogenase Complex?

High ATP causes Pyruvate Kinase to phosphorylate Phosphorylase Dehydrogenase, inactivating it. Then, if low ATP, Pyruvate Phosphatase will hydrolyze that phosphate, turning PDC on again.

At rest, what happens to the Pyruvate Dehydrogenase Complex?

High levels of ATP, Acetyl CoA, and NADH will prompt Pyruvate Kinase to turn off Pyruvate Dehydrogenase Complex, and not proceed Krebs Cycle. When working out, all ATP, NADH and Acetyl CoA will be exhausted, prompting the activation of PD Phosphorylase and hydrolysis of the phosphate that inactivated the Pyruvate Dehydrogenase Complex.

What is the first control site in the Calvin Cycle? What inhibits/activates it?

Isocitrate Dehydrogenase is the first control site. The binding of isocitrate, NAD+, Mg2+ and ADP is cooperative. However ATP is inhibitory, and NADH is as well by taking the space of NAD+.

What happens to Isocitrate along the Citric Acid Cycle? Which enzyme is responsible? What is the yield? Please draw this reaction!

Isocitrate is oxidated into Oxalosuccinate and then decarboxylated into alpha-Ketogluterate. This is done by isocitrate dehydrogenase! This reactions yields the first NADH, an H+ and CO2.

Where does Pyruvate for Aerobic processes?

It is shutted into the mitochondrial membrane by a specific transporter for further processing (Krebs Cycle)

Where does Pyruvate go for Anaerobic Processes?

It remains in the cytoplasm and undergoes Fermentation to yield ethanol.

What happens to the Krebs Cycle when NAD+ and FAD are low?

It stops, there are steps that require NAD+ to proceed.

What is the hydrogen acceptor working with Succinate Dehydrogenase?

It's FAD, unlike the past three oxidations that yielded electron carriers NADH.

Which enzyme is responsible for the oxidation of L-Malate? Where does L-Malate come from? Please draw the reaction.

Malate Dehydrogenase is responsible for the oxidation of Malate into Oxaloacetate. Malate + NAD+ <===> Oxaloacetate + NADH + H+

Which of the coenzymes function as catalytic cofactors, and which ones as stoichiometric?

Stoichiometric: 1. CoA 2. NAD+ Catalytic: 1. Lipoic Acid 2. FAD 3. Thiamine Pyrophosphate

Which enzyme is responsible for the oxidation of Succinate?

Succinate Dehydrogenase

What makes Succinate Dehydrogenase important outside of Krebs Cycle?

Succinate Dehydrogenase is directly associated with the electron-transport chain, the link between Krebs Cycle and ATP formation! FADH2 releases its protons into Coenzyme Q.

What happens to Succinate at the end of the Krebs Cycle?

Succinate is oxidized, hydrated, and oxidized again to regenerate Oxaloacetate.

Which step in the citric acid cycle yields a high-phosphoryl transfer potential molecule?

Succinyl-CoA Synthetase. Which actually creates succinate from Succinyl-CoA, not the opposite.

Where does the Citric Acid Cycle occur?

The Citric Acid Cycle occurs in the inner mitochondrial matrix.

Explain how the Citric Acid Cycle generates energy!

The Citric Acid Cycle oxidizes electron carriers NAD+ and FAD to NADH and FADH2. Then these electron carriers flow through the electron-transport chain to ATP Synthase where they generate a proton gradient that yield, overall, nine molecules of ATP.

What is the structure responsible for bridging glycolysis to the Citric Acid Cycle?

The Pyruvate Dehydrogenase Complex is a large complex responsible for converting Pyruvate into acetyl CoA.

What occurs in the Formation of Acetyl CoA step in Acetyl CoA synthesis? Please draw the reaction.

The acetyl group in acetyllipoamide is transferred to CoA to form Acetyl CoA by Dihydrolipoyl Transacetylase (E2).

How does succinyl-CoA facilitate substrate level phosphorylation?

***The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside disphophate (ADP) and is catalyzed by Succinyl-CoA Synthetase

What are some of the building blocks for other processes given off by the Citric Acid Cycle?

-Porphyrin, Oxaloacetate, Amino Acids and Nucleotide Bases

What are the steps to acetyl CoA conversion from pyruvate?

1. Decarboxylation 2. Oxidation 3. Formation of Acetyl CoA 4. Regeneration of Acetyl CoA

What are the five coenzymes required to synthesize acetyl coenzyme A from pyruvate?

1. Lipoic Acid 2. NAD+ 3. CoA 4. Thiamine pyrophosphate 5. FAD

*** What are the six enzymatic steps of Pyruvate Dehydrogenase Complex ***

1. Pyruvate is decarboxylated at the active site of E1, forming the hydroxy- ethyl-TPP intermediate, and CO2 leaves as the first product. This active site lies deep within the E1 complex, connected to the enzyme surface by a 20-Å-long hydrophobic channel. 2. E inserts the lipoamide arm of the lipoamide domain into the deep 2 channel in E1 leading to the active site. 3. E catalyzes the transfer of the acetyl group to the lipoamide. The acety- 1 lated arm then leaves E1 and enters the E2 cube to visit the active site of E2, located deep in the cube at the subunit interface. 4. The acetyl moiety is then transferred to CoA, and the second product, acetyl CoA, leaves the cube. The reduced lipoamide arm then swings to the active site of the E3 flavoprotein. 5. At the E3 active site, the lipoamide is oxidized by coenzyme FAD. The reactivated lipoamide is ready to begin another reaction cycle. 6. The final product, NADH, is produced with the reoxidation of FADH2 to FAD.

What occurs in the Decarboxylation step of Acetyl CoA synthesis? Please draw the reaction.

1. The carbanion of Thiamine Pyrophosphate combines with Pyruvate to yield = Hydroxyethyl-TPP. Catalyzed by (E1) of the PDC.

In bacteria, how is synthesis of citrate from oxaloacetate and acetyl CoA an important control point?

ATP is an allosteric inhibitor of Citrate Synthase, which increases the Km value and makes less oxaloacetate bound to Acetyl CoA, and so less Citrate is formed.

What is the reagent that all fuel molecules must be converted into to enter the Krebs cycle?

Acetyl CoA

What inhibits the Pyruvate Dehydrogenase Complex?

Acetyl CoA, NADH and ATP inhibit the activty of PDC.

What happens to Alpha Ketogluterate? Please name the enzyme/complex, and draw the reaction that occurs! Also, what's the yield?

Alpha-Ketogluterate Dehydrogenase causes another oxidative decarboxylation. Yields Succinyl CoA, CO2 and NADH again!

What is the second control site in the Calvin Cycle? What inhibits/activates it?

Alpha-Ketogluterate is inhibited by its products: succinyl CoA and NADH, including ATP.

How does Ca2+ affect Pyruvate Dehydrogenase Complex?

Ca2+ activates the Pyruvate Phosphatase enzyme that will hydrolyze any phosphate groups on PDC and activate PDC activity. More Ca2+ more Krebs

Which reaction catalyzes the formation of Citrate?

Citrate Synthase!

Why is isocitrate a convenient site to halt the Citric Acid Cycle?

Citrate can be transported to the cytoplasm, and inhibit phosphofructikinase, stopping glycolysis.

What occurs in the Oxidation step of Acetyl CoA synthesis? Please draw the reaction.

Hydroxyethyl-TPP is oxidized to form an acetyl group, while transferred to a lipoamide (Lipoic Acid) yielding an energy-rich thioester bond in acetyllipoamide. Catalzed by (E1) of the PDC.

How does the cell overcome the large energy of the malate dehydrogenase reaction?

I think that the oxidation and transfer of protons, in combination with catalytic triad stability, helps facilitate this uphill reaction.

When is FAD always the electron acceptor?

In oxidations that remove two hydrogen atoms from a substrate!

How is succinyl synthesis different depending on the organ?

In tissues with large amounts of cellular respiration, (muscle), the ADP-requiring Isozyme dominates. In tissues with anabolic function, GDP-requiring Isozyme dominates.

What is the nucleoside diphosphokinase reaction?

Nucleoside Diphosphokinase catalyzes the Phosphoryl transfer from GTP to ATP GTP +ADP <===> GDP + ATP

What is the first step in the Citric Acid cycle? Please draw the mechanism.

Oxaloacetate reacts with Acetyl CoA via an aldol condensation to form Citryl CoA, then, hydrolysis occurs liberating CoA to yield Citrate!

Where does this inhibition occur?

PD2 and PD3 of PDC

What are flavoproteins?

Proteins tightly associated with FAD of Flavin Mononucleotides.

Which enzyme turns off the E1 component of Pyruvate Dehydrogenase Complex through Phosphorylation? Which enzyme turns it on again?

Pyruvate Dehydrogenase Kinase. Pyruvate Dehydrogenase Phosphatase.

How does Citrate Synthase avoid hydrolysis of Acetyl CoA?

The active site only permits binding of Oxaloacetate, and once bound, it opens another active site for Acetyl CoA where certain bonds are polarized, and the molecule is oriented in such a way to favor condensation from both Acetyl CoA and Oxaloacetate. The active site is also completely closed, meaning water cannot enter, preventing hydrolysis from occurring until condensation is completed.

What must happen to the Citrate molecule before it continues along the Citric Acid Cycle? Why? Please draw this mechanism!

The enzyme Aconitase, bearing an iron group isomerizes the central OH group to the upper carbon. Positions the hydroxyl group to be oxidized and participate in decarboxylation!

Where is the irreversible step in the Citric Acid Cycle?

The formation of Acetyl CoA from Pyruvate Dehydrogenase Complex is an irreversible step! Once it is Acetyl CoA, it must go through 1. Lipid Biosynthesis 2. Citric Acid Cycle

What is the overall function of the citric acid cycle?

The function of the citric acid cycle is the harvesting of high energy electrons form carbon fuels.

What occurs in the regeneration of oxidized lipoamide step after Acetyl CoA synthesis? Please draw the reaction.

The pyruvate dehydrogenase complex cannot function again until it is reset, which requires oxidation of lipoamide. This is done by dihydrolipoyl dehydrogenase (E3). Both electrons in dihydro are transferred to FAD . yielding FADH2, and one proton is transferred to NAD+ and the other is released yielding FAD + and NADH with a free H+ ion.

When you eat, how does your body respond metabolically?

The release of insulin signals hits the receptor, which hit the Phosphatase enzyme responsible for activating the Pyruvate Dehydrogenase Complex to produce ATP from the glycolysis that had been occurring.

How is the synthesis of Citrate favorable with such entropic penalty?

The second reaction, the hydrolysis of Citryl CoA to Citrate powers the uphill reaction of Acetyl CoA to Citryl Coa.

Why must the three steps of acetyl CoA conversion be concerted?

To harness the free energy derived from the rate-limiting step of decarboxylation.