Carbohydrate Metabolism II: Aerobic Respiration

Formation through glycolysis

• Pyruvate enters PDC → makes acetyl-CoA

Pyruvate dehydrogenase

• Pyruvate oxidized → makes CO₂
• 2C molecule binds to TPP (coenzyme held by noncovalent interactions to PDH)
• Mg²⁺ required

Dihydrolipoyl transacetylase

• 2C group bonded to TPP is oxidized + transferred to lipoic acid (bonded coenzyme)
• Disulfide group acts as oxidizing agent → makes acetyl group
• Acetyl group bonded via thioester linkage
• Catalyzes CoA-SH interaction → transfers acetyl group

Dihydrolipoyl dehydrogenase

• FAD used as coenzyme to deoxidize lipoic acid
• Reduces FAD → FADH₂

Other pathways that make acetyl-CoA

• Fatty acid oxidation (cytosol)
  • Activation forms thioester bond between fatty acid carboxyl group and CoA-SH
  • Acyl group transferred to carnitine → crosses inner membrane
  • Crosses membrane, then fatty acyl group transferred back to CoA-SH
  • β-oxidation removes 2C fragments from carboxyl end of acyl-CoA
• Amino acid catabolism
  • AAs lose amino group via transamination
  • Carbon skeletons make ketone bodies
  • When PDC inhibited, ketones can make acetyl-CoA (usually reverse)
• Alcohol
  • Moderate alcohol: alcohol dehydrogenase + acetaldehyde dehydrogenase convert OH to CoA
  • NADH buildup inhibits Krebs cycle
  • Acetyl-CoA made is used to make fatty acids

Key reactions

Citrate formation

• Acetyl-CoA + OAA condense → citryl-CoA
• Hydrolysis → citrate + CoA-SH

Citrate → isocitrate

• Achiral citrate isomerized
• Forms cis-aconitate, then H₂O added → isocitrate

α-KG and CO₂

• Isocitrate oxidized → oxalosuccinate (by isocitrate dehydrogenase)
• Oxalosuccinate decarboxylated → α-KG
• Rate-limiting enzyme: isocitrate dehydrogenase
• Makes first NADH

Succinyl-CoA and CO₂

• Done by α-KGDC (similar to PDC)
• α-KG + CoA → CO₂
• Makes another NADH

Succinate formation

• Hydrolysis of thioester bond on succinyl-CoA → succinate + CoA-SH
• Catalyzed by succinyl-CoA synthetase
• Makes new covalent bonds with energy input
• Thioester bonds in acetyl-CoA release energy when broken
• GDP → GTP phosphorylation driven by thioester hydrolysis energy

Fumarate

• Takes place in matrix (not membrane)
• Succinate dehydrogenase oxidizes succinate → fumarate
• SDH is a flavoprotein (bonded to FAD)
• FAD → FADH₂ because reducing power can’t reduce NAD⁺

Malate

• Fumarase hydrolyzes alkene in fumarate → malate
• Only L-malate forms

OAA made again

• Malate dehydrogenase oxidizes malate → OAA
• Makes another NADH

Net results and ATP yield

PDC

• $\text{Pyruvate} + \text{CoA-SH} + \text{NAD}^+ \rightarrow \text{acetyl-CoA} + \text{NADH} + \text{CO}_2 + \text{H}^+$

Citric acid cycle

• $\text{acetyl-CoA} + 3\text{NAD}^+ + \text{FAD} + \text{GDP} + \text{P}_i + 2\text{H}_2\text{O} \rightarrow 2\text{CO}_2 + \text{CoA-SH} + 3\text{NADH} + 3\text{H}^+ + \text{FADH}_2 + \text{GTP}$

ATP production

• $4(2.5) + 1(1.5) + 1 = 12.5 \text{ per pyruvate} = 25 \text{ per glucose}$
• Glycolysis makes another $7$ $[(2(2.5) + 2)]$ → total 30–32 ATP

PDC regulation

• Pyruvate dehydrogenase kinase
  • When ATP rises → phosphorylates PDH → inhibits acetyl-CoA formation
• Pyruvate dehydrogenase phosphatase
  • Reactivates PDC when ADP high
• High fat fuel:
  • Acetyl-CoA from fats makes carb → acetyl-CoA redundant
  • Why high fat meal fills you up

Control points

• Citrate synthase
  • ATP and NADH allosterically inhibit
  • Citrate directly inhibits citrate synthase
• Isocitrate dehydrogenase
  • Inhibited by ATP and NADH
  • ADP and NAD⁺ activate
• α-KGDC
  • Succinyl-CoA and NADH inhibit
  • ATP inhibits
  • ADP and Ca²⁺ stimulate

E⁻ flow and complexes

• ATP synthesis is endergonic, e⁻ transport is exergonic → coupled

Complex I (NADH–CoQ oxidoreductase)

• Transfers e⁻ from NADH → coenzyme Q (CoQ)
• Contains iron–sulfur clusters + flavoprotein
• NADH transfers e⁻ to FMN:
  • NADH → NAD⁺ and FMNH₂
• Reduced iron–sulfur donates e⁻ from FMNH₂ → CoQ
• Proton pumping: 4 H⁺ moved

Complex II (succinate–CoQ oxidoreductase)

• Transfers e⁻ to CoQ
• Receives e⁻ from succinate:
  • succinate oxidized, FAD → FADH₂
• Reduces iron–sulfur protein and makes FAD
• No proton pumping

Complex III

• Cytochrome reductase
• Transfers e⁻ from CoQ → cytochrome c (steps)
• Cytochromes have heme groups:
  • Fe³⁺ reduced → Fe²⁺
• Proton motive force via Q cycle
• Ubiquinol → ubiquinone
• Moves 4 H⁺

Complex IV

• Transfers e⁻ from cytochrome c → oxygen
• Final proton pumping site
• Cyanide inhibits this

Proton motive force

• ↑ $[H^+]$ → pH drops
• Voltage difference between intermembrane space and matrix increases
• Creates electrochemical gradient
• Called proton motive force because it’s based on protons
• ATP synthase uses this

NADH shuttles

• ATP totals vary because efficiency varies by cell type
• Cytosolic NADH (from glycolysis) can’t cross into matrix → needs shuttles

Glycerol-3-phosphate shuttle

• Cytosolic G3P dehydrogenase oxidizes NADH → NAD⁺ while DHAP → G3P
• On other side, mitochondrial isoform is FAD-dependent:
  • FAD reduced → FADH₂
• Transfers e⁻ to ETC via Complex II

Malate–aspartate shuttle

• Cytosolic OAA can’t cross membrane → reduced to malate
  • Malate dehydrogenase oxidizes NADH → NAD⁺
• Malate enters matrix, then NADH generated in matrix feeds ETC via Complex I
• OAA transaminated → aspartate
• Aspartate enters cytosol → reconverted to OAA

Chemiosmotic coupling

• ATP synthase spans membrane:
  • F₀ portion = ion channel
  • Protons move down gradient into matrix
• Coupling = using proton gradient energy to make ATP
• F₁ portion uses released energy to phosphorylate ADP

Conformational coupling

• Proton gradient and ATP synthesis are indirect
• F₁ portion acts like a turbine (spins)

Regulation

• O₂ is limited → oxidative phosphorylation decreases
• $[\text{NADH}]$ and $[\text{FADH}_2]$ increase
• NADH accumulation inhibits cycle
• Respiratory control can affect pathways