Biochemistry I Fall Term, 2000

Lecture 32: Citric Acid Cycle

Assigned reading in Campbell: Chapter 15

Take the Review Quiz on Lecture 32 concepts.

Citric Acid Cycle Intermediates: Chime images of the reactions in the pathway.
Metabolic Pathway Simulation and the effects of an inhibitor
Citric Acid Cycle Summary Class handout on the reactions in the pathway.

Some General Features

• Sir Hans Krebs, who won a Nobel Prize for its discovery, preferred the term "Tricarboxylic Acid Cycle" (TCA cycle).
• The enzymes that participate in the citric acid cycle are found in the mitochondrial matrix.
• The pathway is amphibolic, i.e. the reactions are involved in catabolic (breakdown) and anabolic (biosynthesis) processes.
• In contrast to glycolysis, none of the intermediates are phosphorylated; but all are either di- or tricarboxylic acids.
• In contrast to the elaborate allosteric regulation of glycogen metabolism, most of the regulation is provided by substrate availability and product inhibition.

Four Key Enzymes in the Citric Acid Cycle

1. Pyruvate Dehydrogenase Complex (PDH) "step O".

• Oxidative decarboxylation produces CO₂ and NADH.
• The acetyl-CoA product is a ("high-energy") thioester.
• The structure of the "complex" is complex (Fig. 15.4).
• The four separate reactions are complex (Fig. 15.3).
• Note the "swinging arm" of reactions 2-4.
• Regulation
  1. ATP and NADH are inhibitors.
  2. Phosphorylation of PDH inhibits the reaction.
  3. Ca²⁺ activates the dephosphorylation.

2. Citrate Synthase: step 1.

• Irreversible condensation reaction.
• Allosteric inhibitors: ATP and NADH.
• Recall that citrate (in the cytosol) is an activator of PFK in glycolysis.

3. Isocitrate Dehydrogenase: step 3.

• Oxidative decarboxylation produces CO₂ and NADH.
• The a-ketoglutarate product is a 5-carbon dicarboxylic acid.
• Regulation
    Allosteric inhibitors: ATP and NADH.
    Allosteric activators: ADP and NAD⁺.

4. a-Ketoglutarate Dehydrogenase Complex: step 4.

• Oxidative decarboxylation produces CO₂ and NADH.
• The succinyl-CoA product is a thioester.
• The complex is similar to the PDH complex.

The remaining reactions in the cycle (steps 5-8) serve to regenerate oxaloacetate for step 1. Some highlights:

1. 1 FADH₂ and 1 NADH are produced.
2. GTP formation (step 5) is a substrate-level phosphorylation.
   Recall 3-PGA dehydrogenase in glycolysis.
   [A phosphohistidine intermediate in the reaction convinced many biochemists in the 1950's that all of oxidative phosphorylation was due to chemical coupling instead of chemiosmotic coupling.]
3. Succinate dehydrogenase is an integral membrane protein.

Three recommended "laps around the cycle" (focusing on the key enzymes):

Fig. 15.6: the reactions

Table 15.2: the energetics.

Fig. 15.7: the regulation.

Study guide: Can you associate the main points of the above three summaries to the schematic diagram on the lecture handout?

[Section 15.5, The glyoxylate cycle, will not be covered.]

The above outline emphasized pyruvate as the entry compound to the catabolic function of the citric acid cycle. This standard description provides a tidy transition from glycolysis. In fact, the citric acid cycle is central to nearly all catabolic pathways. As shown in Campbell's Fig. 15.9, most of the amino acids, sugars and lipids we have studied can be oxidized in the cycle, following their conversion to the 2-, 3-, 4-, or 5-carbon intermediates on the pathway.

Anabolic Functions of the Citric Acid Cycle

Many of the catabolic reactions leading to the cycle are reversible, e.g. the amino acid transaminases. Thus, if the cell is actively synthesizing protein, the cycle participates in biosynthesis (anabolism) by providing precursors to the required amino acids. Depletion of oxaloacetic acid under these conditions is prevented by its net synthesis in the pyruvate carboxylase reaction. Recall Lecture 31 on gluconeogenesis where pyruvate carboxylase provided OAA in cells actively synthesizing carbohydrate. Most of the OAA destined for gluconeogenesis (in the cytosol) leaves the mitochondrion as malate. In the cytosol, malate is reconverted to OAA + NADH and then to PEP. This indirect "shuttle" route is required because neither NAD⁺ nor NADH can be transported across the mitochondrial membranes.

The pathway for fatty acid synthesis in the cytosol requires a similar shuttle system involving transport of malate and citrate out of the mitochondrion. If the available (and storage) supply of carbohydrate is sufficient, the malate is converted to pyruvate + CO₂ + NADPH in the malic enzyme-catalyzed reaction. This variant of the malate shuttle is significant because the NADPH is used as reducing agent in the fatty acid synthesis pathway.

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