Chapter 11: Citric Acid Cycle

How many ATP molecules can 1 molecule of glucose produce?

32, assuming aerobic conditions & all additional pathways are available

In glycolysis, what does 1 molecule of glucose convert to?

2 molecules of pyruvate, 2 NADH, & 2 ATP

What can 1 NADH molecule can converted to?

2.5 ATP

How much ATP total goes glycolysis produce?

7 (2 direct & 5 through NADH & oxidative phosphorylation)

How much does each pyruvate molecule generate?

12.5 ATP per molecule

What are the 3 cellular respiration major stages?

1. Small fuel molecules (glucose, fatty acids, & some amino acids) are oxidized to acetyl CoA

2. Acetyl CoA enter CAC (Krebs or tricarboxylic acid cycle) & is oxidized to CO₂. Energy is released in form of NADH, FADH₂, & GTP

3. Reduced coenzymes, NADH & FADH₂, are oxidized. These are transferred to O₂, final electron acceptor. Released energy is used to make ATP in oxidative phosphorylation

What are the other byproducts in the first stage besides acetyl CoA?

• NADH from NAD+

• CO₂

What are key points from the second stage?

• 1 molecule of Acetyl CoA produces 10 ATP (1 GTP= 1, 3 NADH= 7.5 ATP, 1 FADH₂= 1.5 ATP)

• No net input for gluconeogenesis

What happens in the conversion of pyruvate to Acetyl CoA?

• Accomplished by a large multienzyme complex known as pyruvate dehydrogenase (PDH) complex

• Substrates: Pyruvate & CoA-SH & NAD+

• Products: NADH & CO₂ & acetyl CoA

• Cofactors: TPP, lipoate, & FAD are present

• No bypass is available

• Truly irreversible step

What are the stoichiometric factors in the conversion?

CoA-SH & NAD+

What are the regenerated cofactors in the conversion?

• Thiamine Pyrophosphate (TPP)

• Lipoic acid (lipoate)

• FAD

What is the 5 step sequence reaction?

• 1: Requires TPP & decarboxylate pyruvate

• 2: Requires lipoic acid & regenerates TPP

• 3: Requires CoA-SH, forms acetyl CoA, leaves lipoic acid in reduced form

• 4: Requires FAD & regenerates oxidized form of lipoic acid

• 5: Requires NAD, regenerates FAD, & generates NADH & H+

What is a co-factor or coenzyme?

Non-peptide molecule that acts together w/ an enzyme to carry out the required biochem reaction. Generally possess functional groups &/or chemical properties which are not present on amino acid side chains

What is the key feature of thiamine pyrophosphate (TPP)?

• Carbon atom b/w nitrogen & sulfur atoms in thiazole ring is much more acidic than most -C- groups

• Can ionize to form carbanion, which readily attacks carbonyl group of pyruvate

• Positively charge nitrogen can act as electron sink to stabilize formation of negative charge & decarboxylation of pyruvate

  • CO₂ released & resonance

What is Vitamin B₁ used for?

Decarboxylation reactions & transketolase

What is key feature of Coenzyme A (CoA)?

Reactive thiol (-SH) group that is essential to CoA’s ability to act as acyl carrier in many reactions

What is Acyl CoA?

• Has acyl group

• Thio-ester

What is CoA-SH?

Acts as carrier of acyl groups

What does lipoic acid (lipoate) act as?

Prosthetic group (tightly or covalently bound coenzyme)

What is a key feature of lipoic acid (lipoate)?

Disulfide bond which undergoes oxidation/reduction reactions & which can also serve as an acyl carrier

What is present in the oxidized form of lipoic acid?

• Disulfide bond

• Covalently bound to Lysine

• Very flexible chain

What is present in the reduced form of lipoic acid?

2 -SH groups

What is present in the acetylated form?

Acyl group & -SH group

What are the 3 specific enzymes of the pyruvate dehydrogenase (PDH) complex?

1. Pyruvate dehydrogenase (E₁)

2. Dihydrolipoyl transacetylase (E₂)

3. Dihydrolipoyl dehydrogenase (E₃)

What are the oxidized & reduced pairs?

OX/RED

• NAD+/NADH

• NADP+/NADPH

• FAD/FADH₂

What are the advantages of a multienzyme complex (PDH)?

• All enzymes are clustered & allow intermediates to be efficiently transferred from 1 enzyme to another w/o diffusing away

What was the 5-step reaction sequence an example of?

Substrate channeling

What does channeling prevent?

Loss of intermediates to other enzymes that also need it

What is the key aspect of the multienzyme complex (PDH)?

Tethered lipoyllysine group

What are most steps in pyruvate to acetyl CoA?

Oxidation/reduction

What does the enzyme in CAC, alpha-ketoglutarate dehydrogenase, use the exact same co-factors & mechanisms to form?

Succinyl CoA

What are the structure of similar?

Pyruvate & alpha-ketoglutamate

What is the formation of Acetyl CoA from pyruvate in humans in metabolism?

Irreversible step

• Human body cannot convert pyruvate to glucose or acetyl CoA to glucose

What are the 3 major fates of acetyl CoA?

• Oxidation by CAC (produces energy (ATP))

• Biosynthesis of lipids (i.e. fatty acids, cholesterol, etc. (building blocks for other biomolecules))

• Acetylation reactions in biosynthetic schemes

What does enzymatic control involve?

• Allosteric binding of product feedback ratios

  • ATP/ADP, ATP/AMP, Acetyl CoA/CoA-SH, NADH/NAD+

• Reversible phosphorylation of a Serine residue in E₁

• Increased [pyruvate], the substrate, causes: inhibition of kinase (allows enzyme to be active)

• Hormones (vasopressin, epinephrine) & alpha-adrenergic agonists (norepinephrine) stimulate pyruvate dehydrogenase complex by: activating phosphatase

• Increase [Ca++] causes: stimulation of phosphatase (allows it to be active)

• Insulin (hormone that favors storage of fuel sources): pyruvate→acetyl CoA→stored as fatty acid (Inc. [fatty acids]→ Dec. PDH complex)

What happens with the increase in ratios w/ ATP, Acetyl CoA, & NADH being higher?

Allosterically activates a kinase

What happens with the decrease in ratios w/ ATP, Acetyl CoA, & NADH being lower?

Allosterically activates a phosphatase

How many steps does the CAC contain?

8

What step directly produces GTP (which can convert to ATP)?

Step 5 (substrate level phosphorylation)

What 4 steps (that all involve dehydrogenase enzymes) directly produce NADH or FADH₂?

Steps 3, 4, 6, & 8

What 2 steps “set-up” oxidation reactions?

Steps 2 & 7

How many carbon atoms enter the CAC & how many leave?

2 enter & 2 leave

• No net input for gluconeogenesis

How can oxaloacetate convert to glucose?

PEP

How many of the first 4 reactions are irreversible, making first half of CAC only operative in 1 direction?

3

What is step 1 of the CAC?

Formation of Citrate

• Acetyl CoA + Oxaloacetate → citrate

• Citrate synthase needed

• H₂O → CoA-SH

• Hydrolysis of thioester of acetyl CoA (Releases energy from high-energy bond)

• Regeneration of CoA-SH (can return to PDH complex or other pathway)

What reaction is step 1?

Aldol condensation

What is step 2 of CAC?

Isomerization of Citrate to Isocitrate

• Citrate → cis-Aconitate → isocitrate

• Aconitase needed

• First step- H₂O leaves, second- H₂O returns

• Readily reversible

• 1 of 2 “set-up” steps

  • Hydroxyl group moves from 1 carbon to adjacent

  • Couldn’t be oxidized before this movement due to lacking hydrogen

What reaction is step 2?

Dehydration/hydration

What is step 3 of CAC?

Oxidation of Isocitrate to alpha-Ketoglutarate & CO₂

• Isocitrate → Oxalosuccinate → alpha-Ketoglutarate

• Isocitrate dehydrogenase required

• NADP+ → NADPH + H+, CO₂ released

• Unstable intermediate formed: beta-keto acid

• 1 carbon was oxidized to CO₂

• Depending on situation, NADH or NADPH can produce

• Alpha-Ketoglutarate is an entry/exit point for CAC intermediates

What reaction is step 3 of CAC?

Oxidative decarboxylation

What is step 4 of CAC?

Oxidation of alpha-Ketoglutarate to Succinyl-CoA & CO₂

• Alpha-Ketoglutarate → Succinyl Co-A

• Alpha-ketoglutarate dehydrogenase complex needed

• Stoichiometric cofactors- CoA-SH & NAD+ (which produces NADH)

• Requires thiamine PP, lipoic acid, & FAD as regenerated cofactors

• Mechanism is identical to PDH complex

• At this point in cycle, Acetyl CoA added 2 carbon atoms & they were oxidized & released as CO₂

What reaction is step 4?

Oxidative decarboxylation

What do steps 5-8 serve to regenerate in CAC?

Oxaloacetate

What else is up with steps 5-8 of CAC?

• All readily reversible

• Also parts of other pathways & used to connect CAC w/ other pathways

What is step 5 of CAC?

Conversion of Succinyl-CoA to Succinate

• Succinyl-CoA → Succinate

• Syccinyl-CoA synthetase required

• GDP + P_i → GTP + CoA-SH

• Substrate level phosphorylation

• High energy thioester bond is transferred to GDP

• Reaction is readily reversible & enzyme is named for reveres reaction

What can GTP be converted to ATP by?

Enzyme nucleoside diphosphate kinase (phosphoryl transfer)

ADP + GTP →← ATP + GDP

What does step 5 require that takes the phosphate in the reaction?

Histidine

What reaction is step 5 of CAC?

Substrate-level phosphorylation

What are steps 6-8 very similar to?

Steps 1-3 in fatty acid oxidation (beta-oxidation)

What are steps 6 & 7 of CAC highly?

Stereospecific

What is step 6 of CAC?

Oxidation of Succinate to Fumarate

• Succinate → Fumarate

• Succinate dehydrogenase required

• FAD → FADH₂

• Readily reversible (shown by delta G)

• FAD is used instead of NAD+

  • Free energy change is insufficient to reduce NAD+

What is typically used to oxidize?

NAD+

What reaction is step 6 of CAC?

Dehydrogenation

What is step 7 of CAC?

Hydration of Fumarate to Malate

• Fumarate → transition state → Malate

• Fumarase required

• OH- & H+ put in

• Hydration is highly stereospecific (only 1 enantiomer is formed)

• “Set-up” step for final oxidation- adds H₂O across double bond

What reaction is step 7?

Hydration

What is step 8 of CAC?

Oxidation of Malate to Oxaloacetate

• L-Malate → Oxaloacetate

• L-Malate dehydrogenase required

  • Same enzyme used in gluconeogenesis

• NAD+ → NADH + H+

• Oxaloacetate was regenerated & cycle was ready to process another acetyl CoA molecule

• Equilibrium of reaction lair far to left under standard thermodynamic conditions

  • Drives forward due to oxaloacetate continually removed by citrate syntahse (high exergonic)

What reaction is step 8 of CAC?

Dehydrogenase

What are the 3 key transamination reactions that link CAC w/ amino acids?

• alpha-ketogluerate → glutamate (Glu)

• Oxaloacetate → aspartame (Asp)

• Pyruvate → alanine (Ala)

What do anaplerotic reactions serve to do?

Replenish CAC intermediates if they are diminished

What are anaplerotic reactions?

• Pyruvate + HCO₃- + ATP → (pyruvate carboxylase) → oxaloacetate + ADP P_i

• Phosphoenolpyruvate + CO₂ + GDP → (PEP carboxykinase) → oxaloacetate + GTP

• Pyruvate + HCO₃- + NADPH → (malic enzyme) → Malate + NADP+

What is the role of biotin?

• Prosthetic group of pyruvate carboxylase

• Specialized carrier of CO₂

• Covalently linked to Lysine via covalent amide bond

  • Similar to Lysine binding to lipoic acid in PDH complex

In reaction of pyruvate carboxylase to carboxybiotinyl-enzyme, what happens with biotin?

• Reactions w/ CO2 to form carboxybiotin

• CO₂ is added to catalytic site 1

  • Rotates & moves to site 2 for further steps

• Entire mechanism requires 7 steps

What do the 3 highly exergonic, irreversible steps of CAC act as?

Control sites

What are the major factors governing control for CAC?

• Substrate availability (high [substrate]→ activate enzyme)

• Inhibition by accumulating products

  • Succinyl CoA (terminal product after 3 irreversible steps)

• Allosteric feedback

  • ATP/ADP

  • NADH/NAD+

What does Ca++ do in control of CAC?

Signal for muscle contraction b/c high demand for ATP production