BIOCHEMISTRY AND HUMAN NUTRITION

Module 3. Metabolism

Lesson 18
TCA CYCLE

18.1 Introduction

• The citric acid cycle, also known as the TCA (tricarboxylic acid) cycle or Krebs cycle (after its discoverer in 1937), is used to oxidize the pyruvate formed during the glycolytic breakdown of glucose into CO₂ and H₂O. The cycle is a major energy source in the form of ATP and also produces precursors for many biosynthetic pathways.
• The citric acid cycle operates in the mitochondria of eukaryotes and in the cytosol of prokaryotes. Succinate dehydrogenase, the only membrane-bound enzyme in the citric acid cycle, is embedded in the inner mitochondrial membrane in eukaryotes and in the plasma membrane in prokaryotes.

18.2 The cycle

The cycle forms the central part of a three-step process which oxidizes organic fuel molecules into C_O2 with the concomitant production of ATP.

18.2.1 Step 1 – Oxidation of fuel molecules to acetyl CoA

A major source of energy is glucose which is converted by glycolysis into pyruvate. Pyruvate dehydrogenase (a complex of three enzymes and five coenzymes) then oxidizes the pyruvate (using NAD⁺ which is reduced to NADH) to form acetyl CoA and CO₂. Since the reaction involves both an oxidation and a loss of CO₂, the process is called oxidative decarboxylation.

18.2.2 Step 2 – The citric acid cycle

The cycle carries out the oxidation of acetyl groups from acetyl CoA to C_O2 with the production of four pairs of electrons, stored initially in the reduced electron carriers NADH and FADH₂.

The cycle has eight stages:

1. Citrate (6C) is formed from the irreversible condensation of acetyl CoA (2C) and oxaloacetate (4C) – catalyzed by citrate synthase.
2. Citrate is converted to isocitrate (6C) by an isomerization catalyzed by aconitase. This is actually a two-step reaction during which cis-aconitate is formed as an intermediate. It is the cis-aconitate which gives the enzyme its name.
3. Isocitrate is oxidized to α-ketoglutarate (5C) and C_O2 by isocitrate dehydrognase. This mitrochondrial enzyme requires NAD⁺, which is reduced to NADH.
4. Α-Ketoglutarate is oxidized to succinyl CoA (4C) and C_O2 by the α-ketoglutarate dehydrogenase complex. Like pyruvate dehydrogenase, this is a complex of three enzymes and uses NAD⁺ as a cofactor.
5. Succinyl CoA is converted to succinate (4C) by succinyl CoA synthetase. The reaction uses the energy released by cleavage of the succinyl-CoA bond to synthesize either GTP (mainly in animals) or ATP (exclusively in plants) from Pi and, respectively, GDP or ADP.
6. Succinate is oxidized to fumarate (4C) by succinate dehydrogenase. FAD is tightly bound to the enzyme and is reduced to produce FADH₂.
7. Fumerate is converted to malate (4C) by fumarase; this is a hydration reaction requiring the addition of a water molecule.
8. Malate is oxidized to oxaloacetate (4C) by malate dehydrogenase. NAD⁺ is again required by the enzyme as a cofactor to accept the free pair of electrons and produce NADH.

18.2.3 Step 3 – Oxidation of NADH and FADH2 produced by the citric acid cycle

The NADH and FADH₂ produced by the citric acid cycle are reoxidized and the energy released is used to synthesize ATP by oxidative phosphorylation.

18.3 Energy Yield

• Each of the three NADH molecules produced per turn of the cycle yields three (2.5) ATPs and the single (1.5) FADH₂ yields two ATPs by oxidative phosphorylation.
• One GTP (or ATP) is synthesized directly during the conversion of succinyl CoA to succinate.
• Thus the oxidation of a single molecule of glucose via the citric acid cycle produces 12 (10) ATP molecules.

18.4 Regulation

• Regulation of the cycle is governed by substrate availability, inhibition by accumulating products, and allosteric feedback inhibition by subsequent intermediates in the cycle.
• Three enzymes in the cycle itself are regulated (citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase) and so is the enzyme which converts pyruvate to acetyl CoA to enter the cycle, namely pyruvate dehydrogenase.
• Citrate synthase is inhibited by citrate and also by ATP (the Km for acetyl CoA is raised as the level of ATP rises);
• Isocitrate dehydrogenase is inhibited by NADH and ATP but activated by ADP;
• Α-ketoglutarate dehydrogenase is inhibited by NADH and acetyl CoA (i.e. product inhibition). However, in eukaryotes the enzyme is also controlled by phosphorylation/ dephosphorylation via pyruvate dehydrogenase kinase and a phosphatase.
• Overall, the cycle speeds up when cellular energy levels are low (high ADP concentration, low ATP and NADH) and slows down as ATP (and then NADH2, succinyl CoA and citrate) accumulates.

18.5 Anaplerotic nature of TCA cycle

The intermediates in the cycle provide precursors for many biosynthetic pathways besides being involved in degradation of acetyl CoA.Anabolic reactions that initiate from TCA cycle are:

• Synthesis of fatty acids from citrate
• Amino acid synthesis following transamination of α-ketoglutarate
• Synthesis of purine and pyrimidine nucleotides from α-ketoglutarate and oxaloacetate
• Oxaloacetate can be converted to glucose by gluconeogenesis
• Succinyl CoA is a central intermediate in the synthesis of the porphyrin ring of heme groups

Fig. 18.1 Citric acid cycle