# Lecture 15: Pyruvate Oxidation and the Citric Acid Cycle ## Biochemistry --- ## Learning Objectives By the end of this lecture, students will be able to: 1. Describe the structure, cofactors, and mechanism of the pyruvate dehydrogenase complex 2. List all eight reactions of the citric acid cycle with enzymes and products 3. Calculate the total yield of NADH, FADH2, and GTP per turn of the cycle 4. Identify the regulated steps and their allosteric regulators 5. Explain the amphibolic nature of the citric acid cycle 6. Describe anaplerotic reactions that replenish cycle intermediates --- ## Lecture Content ### I. The Pyruvate Dehydrogenase Complex (PDC) The pyruvate dehydrogenase complex links glycolysis to the citric acid cycle and is located in the **mitochondrial matrix**. Its overall reaction converts pyruvate plus CoA plus NAD+ to acetyl-CoA plus CO2 plus NADH, with a delta-G^0' of -33.4 kJ/mol, making it irreversible. #### Structure of the Complex The PDC is a multienzyme complex containing three enzymes. **E1 (pyruvate dehydrogenase)** decarboxylates pyruvate using thiamine pyrophosphate (TPP). **E2 (dihydrolipoyl transacetylase)** transfers the acetyl group to CoA using lipoic acid and CoA. **E3 (dihydrolipoyl dehydrogenase)** regenerates oxidized lipoamide using FAD and NAD+. Five coenzymes are required, four of which derive from vitamins: TPP (from thiamine, vitamin B1), lipoic acid (lipoamide), CoA (from pantothenic acid, vitamin B5), FAD (from riboflavin, vitamin B2), and NAD+ (from niacin, vitamin B3). Clinically, thiamine deficiency (beriberi) impairs PDC activity, and this pathway is also affected in chronic alcoholism (Wernicke-Korsakoff syndrome). Arsenic poisoning occurs because arsenic binds to lipoamide, inactivating both PDC and alpha-ketoglutarate dehydrogenase. #### Regulation of PDC The most important regulatory mechanism is **covalent modification**. PDH kinase phosphorylates E1 to render it **inactive**, while PDH phosphatase dephosphorylates E1 to restore **activity**. PDH kinase is activated by ATP, acetyl-CoA, and NADH (products signaling sufficient energy) and inhibited by ADP, CoA, NAD+, and pyruvate (signaling the need for more energy). PDH phosphatase is activated by Ca2+ (important in exercising muscle) and insulin. Additionally, acetyl-CoA and NADH directly inhibit PDC allosterically, while CoA and NAD+ activate it. ### II. The Citric Acid Cycle (TCA Cycle / Krebs Cycle) The citric acid cycle takes place in the **mitochondrial matrix** and functions to oxidize acetyl-CoA to CO2 while generating reduced electron carriers (NADH and FADH2) and GTP. The net reaction per acetyl-CoA is: Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O yields 2 CO2 + 3 NADH + FADH2 + GTP + CoA. ### III. The Eight Reactions **Step 1 (Citrate Synthase)** condenses acetyl-CoA with oxaloacetate and water to produce citrate and CoA-SH. This is irreversible (delta-G^0' = -32.2 kJ/mol) and is regulated by inhibition from ATP, NADH, succinyl-CoA, and citrate. **Step 2 (Aconitase)** reversibly isomerizes citrate to isocitrate via a cis-aconitate intermediate through sequential dehydration and rehydration. Aconitase contains an iron-sulfur cluster ([4Fe-4S]) and also functions as an iron status sensor in its cytoplasmic form (IRP1). **Step 3 (Isocitrate Dehydrogenase)** performs the **first oxidative decarboxylation**, converting isocitrate to alpha-ketoglutarate while releasing the first CO2 and producing the first NADH. This is **irreversible** and a major regulatory step, allosterically activated by ADP and Ca2+ and inhibited by ATP and NADH. **Step 4 (Alpha-Ketoglutarate Dehydrogenase Complex)** carries out the **second oxidative decarboxylation**, converting alpha-ketoglutarate to succinyl-CoA while releasing the second CO2 and producing the second NADH. This complex is structurally and mechanistically similar to PDC, requiring the same five coenzymes. It is activated by Ca2+ and inhibited by succinyl-CoA, NADH, and ATP. **Step 5 (Succinyl-CoA Synthetase)** converts succinyl-CoA to succinate while generating GTP through **substrate-level phosphorylation**, driven by the thioester bond energy of succinyl-CoA. GTP is equivalent to ATP via nucleoside diphosphate kinase. Some tissue-specific isozymes use ADP instead of GDP. **Step 6 (Succinate Dehydrogenase / Complex II)** oxidizes succinate to fumarate using FAD (which is covalently bound) rather than NAD+, because the energy change is insufficient to reduce NAD+. This is the only TCA cycle enzyme embedded in the inner mitochondrial membrane, where it also functions as Complex II of the electron transport chain. Malonate is a competitive inhibitor (structural analog of succinate). **Step 7 (Fumarase)** stereospecifically hydrates fumarate to produce only L-malate. **Step 8 (Malate Dehydrogenase)** oxidizes L-malate to oxaloacetate, producing the third NADH. Although delta-G^0' is +29.7 kJ/mol (unfavorable under standard conditions), the reaction is driven forward by removal of oxaloacetate by citrate synthase in step 1 and by the low [NADH]/[NAD+] ratio maintained by the electron transport chain. A circular diagram of the citric acid cycle showing all eight reactions. Each step shows the substrate, product, enzyme name, and cofactors involved. Carbon atoms are tracked through the cycle (4C + 2C = 6C citrate, two decarboxylations back to 4C oxaloacetate). The three NADH-producing steps, the FADH2-producing step, and the GTP-producing step are highlighted with colored arrows. The three irreversible/regulated steps (citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase) are marked with bold borders. Entry of acetyl-CoA and exit of 2 CO2 are clearly shown. A tally of products per turn is listed: 3 NADH, 1 FADH2, 1 GTP, 2 CO2. ### IV. Energy Yield Per acetyl-CoA (one turn of the cycle), the yield is: 3 NADH at approximately 2.5 ATP each = 7.5 ATP; 1 FADH2 at approximately 1.5 ATP = 1.5 ATP; and 1 GTP = 1 ATP, for a **total of approximately 10 ATP equivalents per acetyl-CoA**. Per glucose through complete oxidation (glycolysis + PDC + TCA), the combined yield of approximately 30 to 32 ATP depends on which shuttle system is used for cytoplasmic NADH. ### V. Regulation of the Citric Acid Cycle The cycle is regulated by energy status and substrate availability. High [NADH]/[NAD+] and high [ATP]/[ADP] ratios inhibit the cycle, signaling abundant energy, while high [ADP] and Ca2+ stimulate it, signaling the need for more energy. The three regulated enzymes are citrate synthase (inhibited by ATP, NADH, succinyl-CoA, citrate), isocitrate dehydrogenase (inhibited by ATP, NADH; activated by ADP, Ca2+), and alpha-ketoglutarate dehydrogenase (inhibited by succinyl-CoA, NADH; activated by Ca2+). Calcium signaling elegantly links muscle contraction (Ca2+ release) to increased TCA cycle flux. ### VI. Amphibolic Nature and Anaplerotic Reactions The TCA cycle is **amphibolic**, serving both catabolic and anabolic functions. Cycle intermediates serve as precursors for biosynthesis: citrate is exported to the cytoplasm for fatty acid synthesis; alpha-ketoglutarate gives rise to glutamate and other amino acids; succinyl-CoA feeds into heme synthesis; and oxaloacetate is a precursor for aspartate and gluconeogenesis. When intermediates are withdrawn for biosynthesis, the cycle must be replenished through anaplerotic reactions. #### Anaplerotic Reactions The most important anaplerotic reaction is **pyruvate carboxylase**, which converts pyruvate plus CO2 plus ATP to oxaloacetate. It is activated by **acetyl-CoA**, which signals that OAA is needed to react with accumulating acetyl-CoA, and requires biotin (vitamin B7) as a cofactor. **Glutamate dehydrogenase** converts glutamate to alpha-ketoglutarate. Various **transamination reactions** funnel amino acids into TCA cycle intermediates. The **propionyl-CoA pathway** converts odd-chain fatty acids and some amino acids to succinyl-CoA. A diagram showing the amphibolic nature of the TCA cycle. The central cycle is drawn with arrows indicating the normal flow of carbon. Branching outward from cycle intermediates are arrows showing biosynthetic pathways: citrate to fatty acids, alpha-ketoglutarate to amino acids, succinyl-CoA to heme, oxaloacetate to glucose (gluconeogenesis) and aspartate. Anaplerotic reactions are shown as arrows feeding back into the cycle: pyruvate carboxylase converting pyruvate to oxaloacetate (with acetyl-CoA activation noted), glutamate to alpha-ketoglutarate, and amino acids to succinyl-CoA. The anaplerotic inputs are drawn in a distinct color (blue) to contrast with the biosynthetic outputs (red). ---