Major concepts

1. Why does it matter which coenzymes are prosthetic coenzymes and which are co-substrates?
2. How does TPP assist in decarboxylation by E₁?
3. Why is an oxidation-reduction reaction required for pyruvate dehydrogenase? Which coenzymes are oxidized, and which are reduced?
4. What happens to AcCoA in one cycle of the citric acid cycle? What happens to oxaloacetate in the same cycle? How is that possible, given that citrate is a symmetrical molecule?
5. Which reactions of the citric acid cycle are oxidative decarboxylations? How are they alike, and how are they different?
6. How does α-ketoglutarate resemble pyruvate, and how does α-ketoglutarate dehydrogenase resemble PDH?

Core knowledge
1. What is the overall reaction catalyzed by pyruvate dehydrogenase (PDH)? What is the order in which substrates are bound and in which products are released?
2. What coenzymes are required by each of the three enzymes (E₁, E₂, and E₃) in PDH, and what does each do in the reaction?
3. What are the enzymes of the citric acid cycle? For each, what is the structure of the substrate(s), and what reaction occurs? What is the structure of each product?
4. What high energy coenzymes are produced in the citric acid cycle, and what reaction produces each?
5. What are the intermediates in the reactions catalyzed by citrate synthase, aconitase, and succinyl-CoA synthetase?
6. Which reactions of the citric acid cycle involve oxidation to produce a double bond, hydration of the double bond, and oxidation? How are the two oxidations different?

Pyruvate Dehydrogenase (PDH) Complex = 3 enzymes + 5 coenzymes (many copies of each)
E₁ = pyruvate dehydrogenase requires TPP (prosthetic)
E₂ = dihydrolipoyl transacetylase requires lipoate (prosthetic) and CoA (co-substrate)
E₃ = dihydrolipoyl dehydrogenase requires FAD (prosthetic) and NAD⁺ (co-substrate)

Overall reaction: pyruvate + NAD⁺ + CoA-SH → acetyl-CoA (AcCoA) + CO₂ + NADH + H⁺

Enzyme function and stages of the reaction:
1. E₁ forms a covalent bond between pyruvate and TPP → CO₂ + hydroxyethyl TPP
2. E₂ has three domains:
          a. a lipoyl domain (a long arm) that moves lipoate from one active site to another
         b. a binding domain that connects E₂ with E₁ and E₃
         c. an acyltransferase domain that catalyzes transfer of
              I. hydroxyethyl from TPP to lipoate (semi-reduces lipoate & oxidizes hydroxyethyl)
              II. acetate from hydrolipoate to CoA-SH → dihydrolipoate + AcCoA
3. E₃ catalyzes transfer of 2 H from dihydrolipoate (regenerates lipoate) to FAD and
         transfer of H⁻ from FAD to NAD⁺ → NADH + H⁺

PDH also contains 2 regulatory enzymes: protein kinase + phosphoprotein phosphatase

Citric Acid Cycle

Overall reaction:
AcCoA + 3 NAD⁺ + FAD + GDP + P_i → 2 CO₂ + CoA-SH + 3 NADH + FADH₂ + GTP
This pathway is a cycle that starts and ends with oxaloacetate.

Reactions of the citric acid cycle
Citrate synthase: oxaloacetate + AcCoA + H₂O → citrate + CoA-SH
     a condensation reaction that occurs in 3 steps:
         1. oxaloacetate and then AcCoA bind, and AcCoA → enol-CoA intermediate
              requires Asp and His functioning in acid-base catalysis
         2. Enol-CoA attacks the keto group of oxaloacetate, with a 2^nd His functioning as HA
              produces citroyl-CoA
         3. H₂O adds to the citroyl-CoA bond → citrate + CoA-SH
Aconitase: citrate → isocitrate
     an isomerization reaction that occurs by reversible dehydration
     involves stereospecific binding of citrate to produce a specific isocitrate
         always leaves the acetate group from AcCoA unchanged, indicating that
         citrate synthase orients condensation to make citrate a pro-chiral molecule

Isocitrate dehydrogenase: isocitrate + NAD + → α-ketoglutarate + CO₂ + NADH + H⁺
     an oxidative decarboxylation reaction that requires Mn²⁺ to stabilize an intermediate

α-Ketoglutarate dehydrogenase:
      α-ketoglutarate + CoA-SH + NAD⁺ → CO₂ + succinyl-CoA + NADH + H⁺
      the second oxidative decarboxylation
     This enzyme is a complex like PDH, so it also requires TPP, lipoate, and FAD.

Succinyl-CoA synthetase: succinyl-CoA + GDP + P_i ↔ succinate + CoA-SH + GTP
     a substrate level phosphorylation that involves phosphorylated His as an intermediate:
     1. Succinyl-CoA + P_i + active site His ↔ CoA-SH + succinyl-P + active site His
     2. Succinyl-P + active site His ↔ succinate + His-P
     3. His-P + GDP ↔ active site His + GTP
     Enzyme is named for the reverse reaction (GTP + succinate ↔ GDP + succinyl-CoA).

Succinate dehydrogenase: succinate + FAD ↔ fumarate + FADH₂
     Dehydrogenation of –CH₂–CH₂– by FAD (a prosthetic group)
     FAD is then regenerated by the co-substrate coenzyme Q (ubiquinone)

Fumarase: fumarate + H₂O ↔ malate
     hydration reaction

Malate dehydrogenase: malate + NAD + ↔ oxaloacetate + NADH + H⁺
     dehydrogenation of CH–OH → C=O and regeneration of oxaloacetate

Regulation of PDH and the citric acid cycle
PDH allosteric regulation:
          activation by AMP, CoA-SH, and NAD⁺ (Ca²⁺)
          inhibition by ATP, AcCoA, NADH
PDH covalent regulation:
      kinase phosphorylates and inactivates E₁, which is reversed by the phosphatase
      ATP allosterically activates the kinase
      This is the exception to the "phosphorylation makes energy available" rule, so
          learn it last (I'm not going to ask a question about it.)
      Also an exception to covalent regulation being triggered by a hormone.
Allosteric regulation of the citric acid cycle enzymes that catalyze irreversible reactions:
     activation by ADP (Ca²⁺ in muscle)
     inhibition by products: NADH, succinyl-CoA, ATP

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