The Citric Acid Cycle Explained

Core Concepts

In this article, you will learn about the steps, mechanism, and purpose of the reactions in the citric acid cycle.

Topics Covered in Other Articles

• Glycolysis
• Oxidation and Reduction
• Condensation Reactions
• Photosynthesis
• E2 Reactions

Introduction

The citric acid cycle, also known as the tricarboxylic acid cycle or the Krebs cycle, is a catabolic aerobic process that living organisms use to generate ATP. In this article, you will learn the eight reactions in the citric acid cycle, how those reactions work, and the products and reactants involved. Throughout the article the terms citric acid cycle, tricarboxylic acid cycle (TCA), and Krebs cycle will be used interchangeably. 

The purpose of the citric acid cycle is to generate energy by oxidizing acetyl-CoA into carbon dioxide and serves as a precursor to other biosynthetic reactions. This process takes place in the matrix of the mitochondria. It captures the energy stored in the thiol ester bond in acetyl-CoA and later conserves it into energy packets of NADH. Although oxygen is not consumed, the cycle is aerobic since NADH and FADH₂ transfer electrons to an oxygen-dependent pathway.

Vocabulary 

• Aerobic respiration: a chemical process used to generate energy from carbohydrates in the presence of oxygen.
• Decarboxylation: removal of a carboxyl group and later release of CO₂.
• Dehydrogenase: an enzyme that facilitates the transfer of electrons in the citric acid cycle
• Hydration: addition of a water molecule.

Products of the Citric Acid Cycle

• 3 NADH molecules
• 2 CO₂ molecules
• 1 FADH₂ molecule
• 1 GTP molecule

Note: Each glucose molecule produces two molecules of acetyl-CoA; therefore, the products listed must be multiplied by two. For example, two molecules of glucose will produce the following:

• 12 NADH molecules
• 8 CO₂ molecules
• 4 FADH₂ molecules
• 4 GTP molecules

Substrates in the Citric Acid Cycle

• Acetyl-CoA
• Citrate
• Isocitrate
• Alpha-ketoglutarate
• Succinyl-CoA
• Succinate
• Fumarate
• Malate
• Oxaloacetate

Enzymes in the Citric Acid Cycle

• Citrate synthase
• Aconitase
• Isocitrate dehydrogenase
• Alpha-ketoglutarate dehydrogenase
• Succinyl-CoA synthetase
• Succinate dehydrogenase
• Fumarase
• Malate dehydrogenase

The Preparation Step: Pyruvate Dehydrogenase Complex

Pyruvate is synthesized into acetyl-CoA through the pyruvate dehydrogenase complex. This multi-enzyme complex, therefore, links glycolysis to the Krebs cycle. PDH complex has three enzymatic active subunits which are: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Importantly, pyruvate dehydrogenase has an allosteric site, which can consequently be regulated with ATP or NADH. Obviously, this step is a crucial form of regulation as it allows living organisms to assess their ATP and AMP levels.

Overview

Positive Modulators	Negative Modulators
AMP	ATP
ADP	Acetyl-CoA

First, with the help of E1, the pyruvate combines with thiamine pyrophosphate (TPP), an enzyme cofactor, to subsequently undergo decarboxylation, in a mechanism similar to a Claisen Condensation. This therefore results in the formation of a hydroxyethyl-TPP intermediate.

The hydroxyethyl group of TPP then attacks the lipoamide sulfide group of E2 and eliminates the TPP derivative. Once the TPP derivative is released, the hydroxyethyl carbanion is oxidized to an acetyl group. Notice, that this step also regenerates TPP for future pyruvate reduction.

As a result of transesterification, the acetyl group is transferred to CoA to produce acetyl-CoA.

E3 re-oxidizes dihydrolipoamide to regenerate the original lipoamide group of E2.

Remember: in a reduction reaction, there is a gain of electrons, and in oxidation, there is a loss of electrons. The electrons lost from E3 in this reaction assist in the production of NADH, which helps in the production of ATP during the electron transport chain. As is evident, the pyruvate dehydrogenase complex does not directly produce ATP.

Step 1: Citrate Synthase/Acid-Base Reaction

Citrate synthase catalyzes the reaction between an intermediate called oxaloacetate and acetyl-CoA to later form citrate. The mechanism begins with an aldol condensation reaction and ends with the hydrolysis of a thioester bond. However, before the reaction can occur the histidines-274 and -320 of the synthase has to be protonated, while aspartate-375 is deprotonated at rest. 

Mechanism

Step 2: Aconitase/Dehydration-Rehydration Reaction

Aconitase is an iron-sulfur enzyme that interconverts citrate into its isomer, isocitrate, via cis-aconitase. This is a two-step mechanism that involves the addition and later removal of water.

Mechanism

Step 3: Isocitrate Dehydrogenase/Oxidative Decarboxylation

Isocitrate dehydrogenase is an enzyme that catalyzes the oxidative decarboxylation of isocitrate to its isomer alpha-ketoglutarate. Not only is it a key rate-limiting step, but it is also the first step in the cycle that releases NADH and CO₂. Mg²⁺ helps stabilize the carbonyl on the enolate intermediate, which eventually converts to alpha-ketoglutarate. Importantly, this step can be subsequently regulated given the presence of NADH or ATP.

Mechanism

Step 4: Alpha-Ketoglutarate Dehydrogenase

Alpha-ketoglutarate dehydrogenase is a multi-enzyme complex that converts alpha-ketoglutarate to succinyl-CoA through oxidative decarboxylation. Further, this enzyme produces the second NADH and CO₂ of the citric acid cycle. Also, the alpha-ketoglutarate dehydrogenase complex mechanism is similar to that of pyruvate dehydrogenase.

Mechanism

Step 5: Succinyl-CoA Synthetase (Substrate-Level Phosphorylation)

Succinyl-CoA synthetase cleaves the high-energy thiol ester of succinyl-CoA to form succinate. The energy released from the thioester bond of succinyl-CoA is used to generate GTP, which can then transfer its phosphate to ADP to yield ATP. The phosphate does not transfer directly to the GTP from the succinyl-phosphate, but rather through a histidine residue in the synthetase.

Mechanism

Step 6: Succinate Dehydrogenase (Stereospecific Dehydration)

Succinate dehydrogenase catalyzes the dehydrogenation reaction of succinate to fumarate. This enzyme contains the prosthetic group, FAD, which is covalently linked through a histidine residue. When succinate is oxidized, two hydrogen atoms are then transferred to FAD, producing FADH₂. Succinate dehydrogenase is the only membrane-bound enzyme of the citric acid cycle and is directly linked to the electron transport chain.

Mechanism

Step 7: Fumarase (Hydration Reaction)

Fumarase catalyzes the hydration reaction of fumarate by adding water across the double bond to produce malate.

Mechanism

Step 8: Malate Dehydrogenase

Malate dehydrogenase couples the further oxidation of malate and reduction of NAD+. This forms a carbonyl, converting malate into oxaloacetate, finally completing the citric acid cycle.

Mechanism

Summary of Reactions in the Citric Acid Cycle