BIOCHEM CH9

Biochemistry II Lecture 9 Study Notes

The Citric Acid Cycle (CAC)

Overview of the Pyruvate Dehydrogenase (PDH) Complex

  • The PDH complex contains multiple copies of three key enzymes:

    • Pyruvate dehydrogenase (E1)

    • Dihydrolipoyl transacetylase (E2)

    • Dihydrolipoyl dehydrogenase (E3)

  • The structure features an E2 core made up of 24-60 copies that is surrounded by multiple and variable numbers of E1 and E3 copies.

Functional Characteristics of the PDH Complex

  • The PDH complex channels its intermediates through five sequential reactions:

    • Starts with pyruvate (CH3-C-C)

    • Converts pyruvate to hydroxyethyl-TPP using TPP as a cofactor.

    • Involves acetyl-CoA formation as a critical step, producing Acetyl-CoA (CH3-C-S-COA).

    • The reduction of lipoyllysine and the production of NADH and FADH2 as byproducts.

Reactions of the Citric Acid Cycle

  • One oxaloacetate molecule can theoretically oxidize an infinite number of acetyl groups.

  • Energy is conserved during the four oxidations within the cycle as NADH and FADH2.

Regulation of the Citric Acid Cycle

  • The regulation of the citric acid cycle is crucial due to its central role in metabolism. It involves:

    • Both allosteric and covalent regulation mechanisms that overlap to achieve metabolic homeostasis.

    • Some mutations within the cycle can lead to tumor formation.

Points of Regulation in the Cycle

  • Regulation occurs at several crucial enzymatic points:

    • PDH complex

    • Citrate synthase

    • Isocitrate dehydrogenase complex

    • α-Ketoglutarate dehydrogenase complex

Specific Regulation Mechanisms of PDH Complex

  • PDH complex activity can be:

    • Turned off when:

    • There are high levels of fatty acids and acetyl-CoA available as fuel.

    • The [ATP]/[ADP] and [NADH]/[NAD+] ratios are elevated.

    • Turned on when:

    • Energy demands are high, requiring greater flux of acetyl-CoA into the citric acid cycle.

Covalent Modification of PDH Complex

  • PDH Kinase:

    • Inhibits the PDH complex through phosphorylation.

    • It is allosterically activated by products of the complex but inhibited by substrates.

  • PDH Phosphatase:

    • Reverses inhibition caused by PDH kinase.

Regulation at Exergonic Steps

  • The citric acid cycle is further regulated at strongly exergonic steps catalyzed by:

    • Citrate synthase

    • Isocitrate dehydrogenase complex

    • α-Ketoglutarate dehydrogenase complex

  • Flux is affected by the concentration ratios of different substrates and products:

    • End products (ATP, NADH) exert an inhibitory effect.

    • In contrast, NAD+ and ADP have a stimulatory effect.

    • Long-chain fatty acids also have an inhibitory effect.

Regulation Through Metabolite Flow

  • The regulation of metabolite flow through the citric acid cycle, showcasing the roles of various metabolites:

    • Pyruvate - influenced by products (ATP, acetyl-CoA, NADH, fatty acids) and substrates (AMP, CoA, NAD+, Ca2+).

    • Citrate - influenced by ATP and phosphorylated by citrate synthase.

    • Isocitrate - similarly regulated, with various products affecting its flow through the cycle.

    • α-Ketoglutarate - regulated by similar products as those affecting isocitrate.

Metabolons in the Citric Acid Cycle

  • Metabolons defined as integrated multienzyme complexes held together by noncovament interactions, e.g., malate dehydrogenase, citrate synthase, and aconitase likely form a metabolon.

Fatty Acid Catabolism

Basics of Fatty Acids

  • Fatty acids are identified as carboxylic acids featuring long hydrocarbon chains.

  • Generally, fatty acids are even-numbered, with uncommon instances of those under 14 or over 20 carbons.

  • Over half of fatty acid residues in organisms are unsaturated.

  • The double bond configuration is predominantly in the cis form.

Triacylglycerols

  • Consist of three esterified fatty acids, making them triesters of glycerol.

  • Fats and oils are complex mixtures of triacylglycerols, with variance in fatty acid composition derived from the organism's biological sources.

Oxidation of Long-Chain Fatty Acids

  • Serves as a primary energy-yielding pathway in many organisms, contributing up to 80% of the energy needs for mammalian heart and liver tissues and over 40% of daily energy requirements.

  • The electrons extracted from fatty acids during oxidation traverse the respiratory chain to propel ATP synthesis, ultimately leading to complete oxidation of acetyl-CoA in the citric acid cycle.

β-Oxidation of Fatty Acids

  • Definition: β-oxidation refers to the oxidation of the fatty acyl group at the C-3 position, occurring post-activation at the C-1 carboxyl group via coenzyme A attachment.

Pathways for Fatty Acids

  • Metabolites of various origins converge into a few essential pathways, through processes like triacylglycerol metabolism, fatty acid synthesis, oxidation, and mitochondrial functions.

Evolutionary Perspectives

  • Evolution has favored mechanisms that render reactions energetically favorable. This includes the conversion of fatty acids to thioesters, akin to processes seen in the citric acid cycle where carbonyl groups are generated adjacent to CH2 groups for bond breakdown in fatty acid chains.

Digestion, Mobilization, and Transport of Fats

Sources of Fatty Acid Fuels

  • Cells access fatty acid fuels from four distinct sources:

    • Fats consumed via diet

    • Stored fats as lipid droplets in cells

    • Fats synthesized in one organ for transport to another

    • Fats derived from autophagy processes.

Absorption and Transport in the Small Intestine

  • Dietary fats undergo a sequential breakdown process:

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

    • Intestinal lipases degrade triacylglycerols, facilitating nutrient absorption.

    • Breakdown products are absorbed into intestinal mucosa and reformed into triacylglycerols for transport.

    • Chylomicrons are then transported through the lymphatic system and bloodstream to tissues where fatty acids enter cells.

Storage of Excess Fatty Acids

  • In the liver, fatty acids convert into triacylglycerols, which combine with specific apolipoproteins to form very low-density lipoproteins (VLDLs). These are secreted into the bloodstream and transported to adipose tissues for storage in lipid droplets within adipocytes.

Mobilization of Triacylglycerols

  • Mobilization is initiated by glucagon, leading to lipase-mediated degradation of stored fats within adipose tissues, and explaining the transport mechanisms to myocardial cells where energy production occurs.

Human Serum Albumin

  • Free fatty acids released by lipases bind to serum albumin, a protein that accounts for roughly half of total serum protein, ensuring effective transport to target tissues.

Entry of Glycerol into Glycolytic Pathway

  • Glycerol, primarily derived from triacylglycerol breakdown, can enter glycolysis post-phosphorylation by glycerol kinase to form glycerol-3-phosphate.

Activation and Transport of Fatty Acids into Mitochondria

  • Short-chain fatty acids (<12 carbons) traverse mitochondrial membranes freely.

  • Longer-chain fatty acids utilize the carnitine shuttle for mitochondrial entry after being activated to fatty acyl-CoA.

Fatty Acyl-CoA Synthetase

  • Enzyme responsible for activating fatty acids by converting them into fatty acyl-CoA thioesters, confirmed by the reaction:
    ( ext{fatty acid} + ext{CoA} + ext{ATP} ⇌ ext{fatty acyl-CoA} + ext{AMP} + ext{PPi}).

Formation of Fatty Acyl-CoA

  • The process includes:

    • Two-step formation of fatty acyl-CoA derivatives.

    • Hydrolysis of generated pyrophosphate, with overall reaction given by:
      ( ext{fatty acid} + ext{CoA} + ext{ATP} ⇌ ext{fatty acyl-CoA} + ext{AMP} + 2 ext{P}_i ext{ and } ext{ΔG'}° = -34 ext{ kJ/mol}).

Role of Carnitine

  • Carnitine facilitates the transport of fatty acyl-CoAs across the mitochondrial membrane.

Carnitine Acyltransferase 1 (CAT1)

  • Enzyme responsible for attaching carnitine to fatty acyl-CoA, creating a fatty acyl-carnitine complex for mitochondrial entry.

Acyl-Carnitine/Carnitine Cotransporter

  • This transporter mediates the passive movement of acyl-carnitine into the mitochondrial matrix alongside the entry of free carnitine into the intermembrane space.

Carnitine Acyltransferase 2 (CAT2)

  • Transfers the fatty acyl group back to coenzyme A, regenerating fatty acyl-CoA while freeing carnitine for further cycles.

Pools of Coenzyme A

  • There are two pools:

    • One in the cytosol for fatty acid biosynthesis.

    • One in the mitochondrial matrix focused on oxidative degradation pathways.

Control Point in Fatty Acid Oxidation

  • The carnitine shuttle represents a key regulatory point for fatty acid oxidation within the mitochondria, as its activity is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis.