Block 1 Unit 1 TCA Cycle Objectives

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Dr. Manning's Objective Questions about the TCA Cycle on page 302.

Acetyl-CoA + FAD + GDP + Pi + 2H2O + 3NAD+ -> CoA + 2CO2 + FADH2 + GTP + 3NADH + 3H+ ; This reaction takes place in the mitrochondrial matrix.

Write the overall equation for the TCA Cycle. Where does it take place?

acetyl CoA C2 -> citrate C6 -> cis-aconitase C6 -> isocitrate C6 -> a-ketoglutarate C5 -> succinyl CoA C4 -> succinate -> C4 -> fumarate C4 -> malate C4 -> oxeloacetate C4

Starting with acetyl-CoA (C2), what is the number of carbons at each step of the cycle?

citrate synthase

What enzyme catalyzes the conversion of acetyl CoA and oxaloacetate to citrate?

aconitase

What enzyme catalyzes the conversion of citrate to cis-aconitate

aconitase

What enzyme catalyzes the conversion of cis-aconitate to isocitrate?

isocitrate dehydrogenase

What enzyme catalyzes the conversion of isocitrate to a-ketoglutarate?

a-ketoglutarate dehydrogenase complex

What enzyme catalyzes the conversion of a-ketoglutarate to succinyl CoA?

succinyl CoA synthetase

What enzyme catalyzes the conversion of succinyl CoA to succinate?

succinate dehydrogenase

What enzyme catalyzes the conversion of succinate to fumarate?

fumarase

What enzyme catalyzes the conversion of fumarate to malate?

malate dehydrogenase

What enzyme catalyzes the conversion of malate to oxaloacetate?

pyruvate dehydrogenase complex

What enzyme catalyzes the conversion of pyruvate to acetyl CoA?

acetyl CoA + oxaloacetate -> citrate

What reaction does citrate synthase catalyze?

citrate -> cis-aconitate; cis-aconitate -> isocitrate

What reaction(s) does aconitase catalyze?

isocitrate -> a-ketoglutarate

What reaction does isocitrate dehydrogenase catalyze?

a-ketoglutarate -> succinyl CoA

What reaction does the a-ketoglutarate dehydrogenase complex catalyze?

succinyl CoA -> succinate

What reaction does succinyl CoA synthetase catalyze?

succinate -> fumarate

What reaction does succinate dehydrogenase catalyze?

fumarate -> malate

What reaction does fumarase catalyze?

malate -> oxaloacetate

What reaction does malate dehydrogenase catalyze?

pyruvate -> acetyl CoA

What reaction does the pyruvate dehydrogenase complex catalyze?

isocitrate -> a-ketoglutarate and a-ketoglutarate -> succinyl CoA

Which step(s) in the TCA cycle involve the formation of CO2 (ie. The decarboxylation steps)?

isocitrate -> a-ketoglutarate; a-ketoglutarate -> succinyl CoA; malate -> oxaloacetate

Which step(s) in the TCA cycle involve the formation of NADH + H+?

succinate -> fumarate

Which step(s) in the TCA cycle involve the formation of FADH2?

Succinyl-CoA + GDP + Pi → Succinate + GTP → ATP

Which step(s) in the TCA cycle involve the formation of ATP (by substrate level phosphorylation?)

10 ATP are liberated from one turn of the cycle.

The total number of ATP that can be generated from one turn of the cycle (include any from oxidative phosphorylation)?

2.5 ATP

The number of ATP that can be formed from one molecule of NADH.

1.5 ATP

The number of ATP that can be formed from one molecule of FADH2

Isocitrate dehydrogenase and a-ketoglutarate dehydrogenase are inhibited by NADH and ATP.

What enzymes are involved in regulating the TCA cycle?

Isocitrate Dehydrogenase

What enzyme catalyzes the commited step in the TCA cycle?

Isocitrate dehydrogenase is stimulated by ADP, and inhibited by both ATP and NADH. A-KG is inhibited by succinyl CoA, NADH, and ATP.

How are isocitrate dehydrogenase and a-ketoglutarate dehydrogenase turned on and off?

1) Pyruvate + CoA + NAD+ → AcCoA + NADH + H+ + CO2 and 2)αKG + CoA + NAD+ → SuccCoA + NADH + H+ + CO2. The reactions proceed in an analogous manner and require the same five coenzymes (TPP, Lipoic Acid, CoA, FAD, and NAD+).

What are the similarities between the oxidative decarboxylation of pyruvate and a-ketoglutarate?

TPP (Thiamine Pyrophosphate), Lipoic Acid, CoA, FAD, NAD+

Name the 5 coenzymes which participate in the pyruvate → acetyl CoA and α-Ketoglutarate → succinyl CoA steps.

citrate, cis-aconitate, isocitrate

Name the tricarboxylic acids participating in the T.C.A cycle.

anaplerotic reactions

This term is used to describe reactions which form intermediates of the TCA cycle.

There are a few anaplerotic reactions, but the one of most physiological importance is Pyruvate -> Oxaloacetate via the enzyme pyruvate carboxylase.

Which intermediate of the TCA cycle can be replenished by an anaplerotic reaction?

Pyruvate carboxylase catalyzes the reaction and is activated by acetyl-CoA; biotin is the coenzyme that is required in order for pyruvate to be carboxylated.

What enzyme catalyzes Pyruvate -> Oxaloacetate? What regulates this enzyme? What coenzyme is required?

Glutamate can be converted to a-KG via glutamate dehydrogenase.

How can a-ketoglutarate be regenerated? Include precursor and enzyme.

a-ketoglutarate

What intermediate of the cycle can serve as a precursor to glutamate?

oxaloacetate

What intermediate of the cycle can serve as a precursor to aspartate?

citrate

What intermediate of the cycle can serve as a precursor to acetyl CoA-->fatty acids

succinyl-CoA

What intermediate of the cycle can serve as a precursor to heme synthesis?

Biotin is a coenzyme which facilitates the carboxylation of pyruvate. Biotin provides an active form of CO2.

What is the role of biotin in the conversion of pyruvate->oxaloacetate?

The reaction takes place in the mitochondrial matrix. It is important for the metabolism of glucose because it is irreversible and the reaction must proceed into the TCA cycle. Otherwise, pyruvate will be converted to lactic acid.

Where does the following reaction take place and why is it so important in the metabolism of glucose? Pyruvate->AcetylCoA

There is a symport protein carrier that allows pyruvate to cross into the mitochondrial matrix.

How does pyruvate traverse the inner mitochondrial membrane?

Ca+2 activates PDH phosphatase, which dephosphorylates PDH and makes it active. ATP is a substrate for PDH kinase, which phosphorylizes PDH and makes it inactive. PDH is also regulated by product inhibition through acetyl-CoA and NADH. Finally PDH is regulated by the need for adequate amounts of NAD+ and CoA.

How is pyruvate dehydrogenase regulated?

Thiamine is a co-factor for transketolase (in pentose phosphate pathway) and a coenzyme for the decarboxylation of a-keto acids.

In general, what type of reaction(s) does thiamine (Vitamin B1) participate as a co-enzyme?

Beri Beri (Dry or Wet), and Wernicke-Korsakoff syndrome

What diseases results from a deficiency of thiamine?

Measure the ratio of lactic acid to pyruvic acid in the blood after administration of glucose or measure the amount of transketolase present in RBCs. Blood and urinary levels of pyruvic acid are characteristically elevated in thiamine deficiency but the ratio is a better indicator.

Describe a diagnostic test for thiamine deficiency?

The carboxyl group in lipoic acid is important because it binds covalently with amino-Lysine of the enzyme (E2). Forms an R-C=O-NH-Lys-Enz. (Amide Linkage)

State the covalent interaction between lipoic acid and the enzyme dihydrolipoyl transacetylase (E2).

niacin

What vitamin is essential for the biosynthesis of NAD+

riboflavin

What vitamin is essential for the biosynthesis of FAD

pantothenic acid

What vitamin is essential for the biosynthesis of CoA

riboflavin

What vitamin is essential for the biosynthesis of FMN

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