Unit 5: Metabolism II - Subjective Questions

The citric acid cycle (Krebs cycle / TCA cycle) is the central pathway of aerobic metabolism occurring in the mitochondrial matrix. It oxidizes acetyl-CoA to CO while producing reduced coenzymes.

The eight steps:

1. Citrate synthesis — Acetyl-CoA + Oxaloacetate Citrate (enzyme: citrate synthase).
2. Isomerization — Citrate Isocitrate (enzyme: aconitase, via cis-aconitate).
3. First oxidative decarboxylation — Isocitrate -ketoglutarate + CO + NADH (enzyme: isocitrate dehydrogenase).
4. Second oxidative decarboxylation — -ketoglutarate Succinyl-CoA + CO + NADH (enzyme: -ketoglutarate dehydrogenase complex).
5. Substrate-level phosphorylation — Succinyl-CoA Succinate + GTP (enzyme: succinyl-CoA synthetase).
6. Oxidation — Succinate Fumarate + FADH (enzyme: succinate dehydrogenase).
7. Hydration — Fumarate Malate (enzyme: fumarase).
8. Oxidation — Malate Oxaloacetate + NADH (enzyme: malate dehydrogenase).

Energy yield per acetyl-CoA:

• 3 NADH
• 1 FADH
• 1 GTP (ATP)
• 2 CO released

When NADH and FADH are oxidized in the ETC, each turn yields approximately 10 ATP.

The citric acid cycle is regulated primarily at three irreversible, exergonic steps governed by the energy and redox state of the cell.

1. Citrate synthase:

• Inhibited by: ATP, NADH, succinyl-CoA, citrate.
• Availability of substrates (acetyl-CoA and oxaloacetate) also controls activity.

2. Isocitrate dehydrogenase (key control point):

• Activated by: ADP and Ca.
• Inhibited by: ATP and NADH.

3. -Ketoglutarate dehydrogenase complex:

• Inhibited by: succinyl-CoA and NADH (products).
• Activated by: Ca.

General principles:

• High energy charge (high ATP/NADH ratio) slows the cycle.
• High ADP and Ca (signaling active muscle contraction) accelerate it.
• The cycle is thus tightly coupled to the cell's demand for ATP.

An amphibolic pathway is a metabolic pathway that functions in both catabolism (breakdown) and anabolism (biosynthesis).

The citric acid cycle is amphibolic because:

Catabolic role:

• Oxidizes acetyl-CoA (from carbohydrates, fats, proteins) to CO, generating NADH, FADH, and GTP for ATP synthesis.

Anabolic role — intermediates are precursors for biosynthesis:

• Citrate exported to cytosol for fatty acid synthesis.
• -Ketoglutarate glutamate and other amino acids.
• Succinyl-CoA porphyrins / heme synthesis.
• Oxaloacetate aspartate, gluconeogenesis (glucose).

Anaplerotic reactions replenish intermediates withdrawn for biosynthesis, e.g., pyruvate carboxylase converts pyruvate oxaloacetate. This dual function makes the cycle central to metabolism.

The glyoxylate cycle is a modified variant of the TCA cycle that allows organisms to convert acetyl-CoA (fat/2-carbon sources) into carbohydrates, bypassing the two decarboxylation steps.

Location & organisms:

• Occurs in plants, bacteria, fungi, and algae within specialized organelles called glyoxysomes (in plants).
• Absent in animals, which cannot achieve net conversion of fat to glucose.

Key unique enzymes:

1. Isocitrate lyase — cleaves isocitrate glyoxylate + succinate.
2. Malate synthase — condenses glyoxylate + acetyl-CoA malate.

Differences from the citric acid cycle:

This cycle enables germinating seeds to convert stored lipids into sugars needed for growth.

The electron transport chain is a series of membrane-bound carriers in the inner mitochondrial membrane that transfer electrons from NADH/FADH to O, coupling this to proton pumping.

Complex I (NADH-CoQ oxidoreductase):

• Transfers electrons from NADH to ubiquinone (CoQ).
• Contains FMN and Fe-S clusters.
• Pumps 4 H across the membrane.

Complex II (Succinate-CoQ oxidoreductase):

• Transfers electrons from FADH (succinate) to CoQ.
• Contains FAD and Fe-S centers.
• Does NOT pump protons.

Complex III (CoQ-cytochrome c oxidoreductase):

• Transfers electrons from CoQH to cytochrome c via the Q cycle.
• Pumps 4 H.

Complex IV (Cytochrome c oxidase):

• Transfers electrons from cytochrome c to O, forming HO.
• Contains cytochromes a, a and Cu centers.
• Pumps 2 H.

Mobile carriers: Ubiquinone (CoQ) and cytochrome c shuttle electrons between complexes. The net result is a proton gradient (proton-motive force) used to synthesize ATP.

The chemiosmotic theory (Peter Mitchell, 1961) explains how the energy of electron transport is coupled to ATP synthesis.

Core postulates:

• As electrons pass through the ETC complexes, protons (H) are pumped from the mitochondrial matrix into the intermembrane space.
• This creates an electrochemical proton gradient across the inner mitochondrial membrane, called the proton-motive force (PMF).

Proton-motive force has two components:

• — membrane electrical potential.
• — chemical (concentration) gradient.

ATP synthesis:

• Protons flow back into the matrix through the enzyme ATP synthase (Complex V).
• This flow drives conformational changes that catalyze the phosphorylation of ADP:
  

Significance: The theory established that the coupling intermediate is not a chemical bond but an electrochemical gradient, explaining why an intact membrane is essential for oxidative phosphorylation. Mitchell received the Nobel Prize in 1978.

ATP synthase is a molecular machine that synthesizes ATP using the proton-motive force. It has two main components:

F unit (membrane-embedded):

• Forms the proton channel.
• Composed of a, b, and c-ring subunits.
• Proton flow rotates the c-ring.

F unit (matrix-facing):

• Composed of subunits.
• The subunits are catalytic sites for ATP synthesis.
• The central stalk rotates within the head.

Binding change mechanism (Paul Boyer):
The three subunits cycle through three conformational states:

• Loose (L) — binds ADP + P loosely.
• Tight (T) — binds substrates tightly, ATP forms spontaneously.
• Open (O) — low affinity, releases ATP.

Rotational catalysis:

• Proton flow rotates the subunit.
• This rotation induces sequential conformational changes (L T O) in each subunit.
• The energy of the PMF is used mainly for the release of ATP, not its formation.

Boyer and Walker shared the Nobel Prize (1997) for this work.

The complete aerobic oxidation of glucose can be accounted for stage by stage (using modern P/O ratios: NADH 2.5 ATP, FADH 1.5 ATP).

1. Glycolysis (cytosol):

• 2 ATP (net, substrate-level)
• 2 NADH 2 1.5 or 2.5 = 3–5 ATP (depends on shuttle)

2. Pyruvate Acetyl-CoA (link reaction, 2):

• 2 NADH 2 2.5 = 5 ATP

3. Citric acid cycle (2 turns):

• 6 NADH 6 2.5 = 15 ATP
• 2 FADH 2 1.5 = 3 ATP
• 2 GTP = 2 ATP
• Subtotal = 20 ATP

Total (using malate-aspartate shuttle):

Using glycerol phosphate shuttle: cytosolic NADH yields FADH-equivalent, giving ~30 ATP.

Note: Older textbooks quote 38 ATP using P/O values of 3 (NADH) and 2 (FADH).

Both processes generate ATP but differ fundamentally in mechanism.

Summary: Substrate-level phosphorylation is a direct chemical reaction, while oxidative phosphorylation is an indirect, membrane-dependent process powered by electron transport.

Oxidative phosphorylation can be disrupted by two classes of agents:

1. Inhibitors — block electron flow or ATP synthesis:

• Complex I inhibitors: Rotenone, Amytal.
• Complex II inhibitor: Malonate (competitive inhibitor of succinate dehydrogenase).
• Complex III inhibitor: Antimycin A.
• Complex IV inhibitors: Cyanide (CN), Carbon monoxide (CO), Azide (N) — bind cytochrome a.
• ATP synthase inhibitor: Oligomycin — blocks the F proton channel.

2. Uncouplers — dissipate the proton gradient without blocking electron transport, so respiration continues but no ATP is made (energy released as heat):

• 2,4-Dinitrophenol (DNP) — a lipophilic weak acid that carries H across the membrane.
• FCCP — carbonyl cyanide-p-trifluoromethoxyphenylhydrazone.
• Thermogenin (UCP-1) — a natural uncoupling protein in brown adipose tissue that generates heat (non-shivering thermogenesis).

Key difference: Inhibitors stop O consumption; uncouplers increase O consumption while abolishing ATP synthesis.

-oxidation is the mitochondrial pathway that degrades fatty acids by removing two-carbon units (acetyl-CoA) at a time from the carboxyl end.

Activation & transport:

• Fatty acid is activated to fatty acyl-CoA (by acyl-CoA synthetase, uses 2 ATP equivalents).
• Transported into mitochondria via the carnitine shuttle (carnitine palmitoyltransferase I & II).

Four repeating steps of each cycle:

1. Oxidation (dehydrogenation): Acyl-CoA trans--enoyl-CoA + FADH (enzyme: acyl-CoA dehydrogenase).
2. Hydration: enoyl-CoA L-3-hydroxyacyl-CoA (enzyme: enoyl-CoA hydratase).
3. Oxidation: hydroxyacyl-CoA 3-ketoacyl-CoA + NADH (enzyme: 3-hydroxyacyl-CoA dehydrogenase).
4. Thiolysis (cleavage): 3-ketoacyl-CoA + CoA acetyl-CoA + acyl-CoA (shortened by 2 C) (enzyme: thiolase).

Per cycle yield: 1 FADH, 1 NADH, 1 acetyl-CoA. The shortened acyl-CoA re-enters the cycle until fully degraded.

Palmitic acid (16 carbons) is fully oxidized via -oxidation and the TCA cycle.

Number of -oxidation cycles:

• Produces 8 acetyl-CoA, 7 FADH, 7 NADH.

Step-by-step ATP accounting (NADH = 2.5, FADH = 1.5):

From -oxidation:

• 7 FADH 1.5 = 10.5 ATP
• 7 NADH 2.5 = 17.5 ATP

From TCA cycle (8 acetyl-CoA):

• Each acetyl-CoA = 10 ATP 8 10 = 80 ATP

Gross total:

Subtract activation cost (2 ATP equivalents used to form palmitoyl-CoA):

Note: Older values (NADH = 3, FADH = 2) give 129 ATP net.

Oxidation of unsaturated fatty acids:
Unsaturated fatty acids contain cis double bonds that cannot be handled by the standard -oxidation enzymes. Two extra enzymes are needed:

1. Enoyl-CoA isomerase — converts a cis- double bond to the trans- configuration required by enoyl-CoA hydratase (needed for monounsaturated fats).
2. 2,4-dienoyl-CoA reductase — used for polyunsaturated fatty acids; reduces a 2,4-dienoyl intermediate (uses NADPH) to trans--enoyl-CoA, which is then isomerized.

Oxidation of odd-chain fatty acids:

• Normal -oxidation proceeds until a 3-carbon propionyl-CoA is left.
• Propionyl-CoA is converted to succinyl-CoA (a TCA intermediate) via three steps:
  1. Propionyl-CoA carboxylase (biotin-dependent) D-methylmalonyl-CoA.
  2. Methylmalonyl-CoA epimerase L-methylmalonyl-CoA.
  3. Methylmalonyl-CoA mutase (vitamin B-dependent) Succinyl-CoA.

Succinyl-CoA then enters the TCA cycle. Thus, odd-chain fatty acids yield some glucogenic contribution.

Fatty acid biosynthesis occurs in the cytosol and builds palmitate (C) from acetyl-CoA.

1. Transport of acetyl-CoA out of mitochondria:

• Acetyl-CoA combines with oxaloacetate citrate, which is exported to the cytosol.
• Cytosolic citrate lyase regenerates acetyl-CoA.

2. Commitment step:

• Acetyl-CoA carboxylase (biotin-dependent, rate-limiting) converts acetyl-CoA malonyl-CoA.
  

3. Fatty acid synthase (FAS) complex:
A multi-enzyme complex with 7 activities plus an acyl carrier protein (ACP). It repeats a 4-step cycle:

• Condensation (of acetyl and malonyl groups)
• Reduction (uses NADPH)
• Dehydration
• Reduction (uses NADPH)

Each cycle adds 2 carbons; after 7 cycles, palmitate (C) is released.

Sources of NADPH:

• Pentose phosphate pathway (major)
• Malic enzyme reaction.

Overall reaction:

Although superficially reverse processes, -oxidation and fatty acid synthesis differ in almost every respect.

The two pathways are reciprocally regulated — malonyl-CoA (a synthesis intermediate) inhibits CPT-I, preventing simultaneous oxidation.

Ketone bodies are water-soluble fuels produced from acetyl-CoA in the liver mitochondria during prolonged fasting, starvation, or uncontrolled diabetes.

The three ketone bodies:

1. Acetoacetate
2. -Hydroxybutyrate (D-3-hydroxybutyrate)
3. Acetone (volatile, exhaled)

Ketogenesis (formation in liver):

1. 2 Acetyl-CoA Acetoacetyl-CoA (thiolase).
2. Acetoacetyl-CoA + Acetyl-CoA HMG-CoA (HMG-CoA synthase).
3. HMG-CoA Acetoacetate + Acetyl-CoA (HMG-CoA lyase).
4. Acetoacetate -hydroxybutyrate (reduction) or spontaneously acetone.

Ketolysis (utilization in extrahepatic tissues — brain, heart, muscle):

• -hydroxybutyrate acetoacetate.
• Acetoacetate + succinyl-CoA acetoacetyl-CoA (enzyme: thiophorase / succinyl-CoA:acetoacetate CoA transferase).
• Acetoacetyl-CoA 2 acetyl-CoA enters TCA cycle.

Note: The liver cannot use ketone bodies for energy because it lacks thiophorase. During starvation, the brain adapts to use ketone bodies, sparing glucose.

The carnitine shuttle transports long-chain fatty acyl groups from the cytosol into the mitochondrial matrix for -oxidation, since the inner mitochondrial membrane is impermeable to acyl-CoA.

Steps:

1. Carnitine palmitoyltransferase I (CPT-I) — located on the outer mitochondrial membrane; transfers the acyl group from acyl-CoA to carnitine, forming acyl-carnitine.
2. Carnitine-acylcarnitine translocase — moves acyl-carnitine across the inner membrane in exchange for free carnitine.
3. Carnitine palmitoyltransferase II (CPT-II) — on the inner membrane matrix side; regenerates acyl-CoA inside the matrix and releases carnitine.

Why required:

• Acyl-CoA cannot directly cross the inner mitochondrial membrane.
• Only long-chain fatty acids need the shuttle; short/medium-chain fatty acids diffuse freely.

Regulation:

• CPT-I is the rate-limiting enzyme and is strongly inhibited by malonyl-CoA (the first intermediate of fatty acid synthesis).
• This ensures fatty acid synthesis and oxidation do not occur simultaneously — a key point of reciprocal regulation.

Cytosolic NADH (produced in glycolysis) cannot cross the inner mitochondrial membrane and must use shuttle systems.

1. Glycerol Phosphate Shuttle:

• Cytosolic NADH reduces DHAP glycerol-3-phosphate (cytosolic glycerol-3-P dehydrogenase).
• Membrane-bound enzyme (FAD-linked) reoxidizes it, passing electrons to FADH/ubiquinone.
• Yields ~1.5 ATP per cytosolic NADH.
• Predominant in skeletal muscle and brain.

2. Malate-Aspartate Shuttle:

• Cytosolic NADH reduces oxaloacetate malate, which crosses the membrane.
• In matrix, malate oxaloacetate regenerating NADH inside.
• Oxaloacetate is transaminated to aspartate to return to cytosol.
• Yields ~2.5 ATP per cytosolic NADH.
• Predominant in liver, kidney, heart.

The malate-aspartate shuttle is more energy-efficient because it regenerates NADH inside the mitochondrion.

The pyruvate dehydrogenase complex (PDC) links glycolysis to the citric acid cycle by converting pyruvate to acetyl-CoA (the link reaction) in the mitochondrial matrix.

Overall reaction:

Structure — three enzymes:

1. E — Pyruvate dehydrogenase (uses TPP / thiamine).
2. E — Dihydrolipoyl transacetylase (uses lipoic acid, CoA).
3. E — Dihydrolipoyl dehydrogenase (uses FAD, NAD).

Five coenzymes required: TPP, lipoic acid, CoA, FAD, NAD (mnemonic: "Tender Loving Care For Nancy").

Regulation:

• Product inhibition: Acetyl-CoA and NADH inhibit the complex.
• Covalent modification:
  • PDH kinase phosphorylates and inactivates PDC (activated by high ATP, acetyl-CoA, NADH).
  • PDH phosphatase dephosphorylates and activates PDC (stimulated by Ca, insulin).
• Allosteric activation by pyruvate, ADP, Ca.

This irreversible step commits carbon from carbohydrates to oxidation or fat synthesis.

Respiratory Control:
Respiratory control refers to the dependence of the rate of electron transport (and O consumption) on the availability of ADP.

• When ADP is abundant (high energy demand), ATP synthase actively uses the proton gradient, protons flow back into the matrix, and electron transport speeds up.
• When ADP is scarce (high ATP), the proton gradient builds up, back-pressure slows proton pumping, and electron transport is inhibited.
• Thus, [ADP] is the key regulator coupling respiration to cellular energy needs.

P/O Ratio:
The P/O ratio is the number of ATP molecules synthesized (P = phosphate esterified) per pair of electrons transferred to one oxygen atom (½ O reduced).

Accepted values (modern):

• NADH: P/O 2.5 (electrons enter at Complex I, pumping 10 H).
• FADH: P/O 1.5 (electrons enter at Complex II, pumping 6 H).

Significance:

• The lower P/O ratio for FADH reflects that its electrons bypass Complex I, pumping fewer protons.
• The P/O ratio is not a whole number because the same proton gradient also powers ATP export and phosphate transport.