Biochemistry II: Lecture 9 Study Notes

The PDH Complex

• The PDH complex, or Pyruvate Dehydrogenase Complex, contains multiple enzymes, specifically:

  • Pyruvate Dehydrogenase (E1)

  • Dihydrolipoyl Transacetylase (E2)

  • Dihydrolipoyl Dehydrogenase (E3)

• It features a core made up of 24-60 copies of E2 surrounded by variable numbers of E1 and E3 copies.

Intermediate Channeling in the PDH Complex

• The PDH complex channels its intermediates through five reactions, which include:

  1. Pyruvate → Hydroxyethyl TPP

  2. Hydroxyethyl TPP → Acetyl-lipoyllysine

  3. Acetyl-lipoyllysine → Acetyl-CoA

  4. FADH2 formation through reduction of lipoyllysine

  5. NAD+ reduction to form NADH + H+

Reactions of the Citric Acid Cycle

• The citric acid cycle can theoretically oxidize an infinite number of acetyl groups using one molecule of oxaloacetate.

• The energy from the four oxidations is conserved as NADH and FADH2:

  • The energy yield from the cycle is influenced by the concentrations of substrates and products.

Regulation of the Citric Acid Cycle

• The central role of the citric acid cycle necessitates its regulation in coordination with various metabolic pathways.

• Regulation occurs through both allosteric and covalent mechanisms, which work in an interactive way to maintain homeostasis. Certain mutations affecting these reactions can lead to tumor formation.

Key Points of Regulation

• Regulation occurs at several points, including:

  • PDH complex

  • Citrate synthase

  • Isocitrate dehydrogenase complex

  • α-ketoglutarate dehydrogenase complex

Allosteric and Covalent Mechanisms in PDH Complex Regulation

• The activity of the PDH complex is regulated by:

  • Off: When ample fatty acids and acetyl-CoA are available, and when [ATP]/[ADP] and [NADH]/[NAD+] ratios are high.

  • On: When energy demands are high, requiring greater flux of acetyl-CoA into the citric acid cycle.

• PDH Kinase inhibits the PDH complex via phosphorylation:

  • Activated by products of the complex and inhibited by substrates.

• PDH Phosphatase reverses inhibition by PDH kinase.

Regulation at Exergonic Steps of the Citric Acid Cycle

• Regulation occurs at strongly exergonic steps catalyzed by:

  • Citrate synthase

  • Isocitrate dehydrogenase complex

  • α-ketoglutarate dehydrogenase complex

• Fluxes are affected by concentrations of substrates and products:

  • Inhibitors: ATP and NADH

  • Stimulators: NAD+ and ADP

  • Inhibitory effects also come from long-chain fatty acids.

Metabolite Flow Through the Citric Acid Cycle

• Pyruvate dehydrogenase regulation is influenced by:

  • Inhibitors: ATP, acetyl-CoA, NADH, fatty acids

  • Activators: AMP, CoA, NAD+, Ca2+

• Regulation continues for Citrate, Isocitrate, and α-Ketoglutarate processes involving various enzymes and metabolites that flow through the citric acid cycle.

Metabolons

• Metabolons: Integrated multienzyme complexes that interact through noncovalent interactions.

• Examples include a combination of malate dehydrogenase, citrate synthase, and aconitase.

Fatty Acid Catabolism

• Fatty acids are defined as carboxylic acids with long hydrocarbon chains. Characteristics include:

  • Generally even-numbered in carbon length.

  • Rarely comprise < 14 or > 20 carbons.

  • Over half of fatty acid residues in plants and animals are unsaturated, generally featuring cis configuration for double bonds.

Triacylglycerols

• Comprised of three esterified fatty acids.

• Form complex mixtures varying with organisms.

Oxidation of Long-Chain Fatty Acids to Acetyl-CoA

• This pathway serves an essential energy-yielding function, providing:

  • Up to 80% of energy demands in mammalian heart and liver.

  • More than 40% of daily energy requirements.

• Electrons removed during oxidation enter the respiratory chain, facilitating ATP synthesis.

• Acetyl-CoA generated can be fully oxidized to CO2 within the citric acid cycle.

β Oxidation

• Defined as the oxidation of the fatty acyl group at the C-3 (β) position following initial activation.

• The process involves:

  • Attachment of the fatty acid’s carboxyl group to CoA, creating a thioester bond.

Sources of Fatty Acid Fuels

• Cells can source fatty acids from:

  • Dietary fats

  • Stored fats in lipid droplets

  • Fats synthesized for export between organs

  • Fats obtained via autophagy

Digestion and Absorption of Dietary Fats

1. Bile salts emulsify dietary fats, forming mixed micelles in the small intestine.

2. Intestinal lipases break down triacylglycerols.

3. Breakdown products are absorbed by intestinal mucosa and reformed into triacylglycerols, then packaged into chylomicrons and move into the bloodstream.

4. Lipoprotein lipase, activated by apoC-II, processes triacylglycerols back into free fatty acids.

Storage and Mobilization of Fatty Acids

• Excess fatty acids are converted to triacylglycerols, then packaged into VLDLs for export to adipose tissue.

• Stored triacylglycerols within adipocytes can be mobilized via hormonal signals from glucagon leading to:

  • Activation of hormone-sensitive lipase (HSL) to release fatty acids into the bloodstream.

Entry of Fatty Acids into the Glycolytic Pathway

• Most energy from triacylglycerols comes from the three long-chain fatty acids.

• Glycerol kinase processes glycerol, facilitating its entry into glycolysis (converting it to glycerol 3-phosphate).

Transport Mechanism of Fatty Acids into Mitochondria

• Short-chain fatty acids can diffuse freely across membranes, while longer chains require a carnitine shuttle.

• Fatty acyl-CoA Synthetase activates fatty acids to fatty acyl-CoA via ATP:
  $ext{fatty acid} + ext{CoA} + ext{ATP} ⇌ ext{fatty acyl-CoA} + ext{AMP} + ext{PPi}$

Mechanism of Carnitine Shuttle

• Carnitine facilitates the transport of fatty acyl-CoA across mitochondrial membranes.

• Catalyzed by Carnitine Acyltransferase 1 (CAT1), which links a fatty acyl group to carnitine, forming fatty acyl-carnitine.

• Movement occurs via a cotransporter where acyl-carnitine enters the matrix while carnitine moves into the intermembrane space.

• Once in the matrix, Carnitine Acyltransferase 2 (CAT2) transfers the fatty acyl group back to CoA, regenerating fatty acyl-CoA for further oxidation.

Coenzyme A in Fatty Acid Pathways

• Two pools of Coenzyme A exist: one in the cytosol for fatty acid biosynthesis and another in mitochondria for oxidative degradation purposes.

• The carnitine shuttle serves as a control point, where its activity is restricted by malonyl-CoA to prevent concurrent fatty acid synthesis and degradation.