Useful Notes on Tricarboxylic Acid Cycle (With Diagram)

The tricarboxylic acid cycle is also called the Krebs cycle because the major steps of the cycle were worked out in 1937 by Sir Hans Krebs.

At that time, radioactively labeled compounds were not available for biological studies and the cellular site of the reac­tions was not known with certainty.

The experiments performed by Krebs and the logic and reasoning with which his findings were interpreted in order to formu­late the metabolic pathway are recognized as mile­stones in our progressive unraveling of cellular metab­olism.

In 1948 E. P. Kennedy and A. L. Lehninger showed that mitochondria isolated from liver tissue homogenates by differential centrifugation contained the enzyme activities of the Krebs cycle reactions. Krebs received the Nobel Prize in 1953 for his elucida­tion of this most important metabolic pathway.

The Krebs cycle reactions are utilized by the cell to further metabolize a number of products of other re­actions in the cytosol. These can be products as di­verse as amino acids, fatty acids, precursors of nucleic acids, and pyruvic acid. However, the greatest meta­bolic merit of the cycle is its oxidation of pyruvate— the product of carbohydrate metabolism in the cyto­sol. The oxidation of this compound in the TCA cycle provides the reducing power to make significant amounts of ATP. The reactions are shown in Figure 16-20 and are described below.

Oxidation of Pyruvate:

The oxidation of pyruvate is brought about by a set of reactions catalyzed by the pyruvate dehydrogenase complex. This complex contains three enzymes (E₁ pyruvate dehydrogenase; E₂, dihydrolipoyl transacetylase; and E₃, dihydrolipoyl dehydrogenase) and five coenzymes and is located in the matrix of the mitochondrion. The first step is the decarboxylation of the pyruvate to yield CO₂ and an α-hydroxyethyl unit attached through thiamine pyrophosphate (TPP, a coenzyme) to enzyme E₁ that is,

The hydroxyethyl group is then dehydrogenated (oxidized) to form an acetyl group, which is trans­ferred to one of the sulfur atoms of lipoic acid, a coen­zyme of E₂.

The acetyl group is then transferred to coenzyme A, forming acetyl-CoA, which separates from the enzyme complex. One hydrogen from the lipoyl group and one from coenzyme A are transferred to flavin adenine dinucleotide (FAD) (Fig. 3-11% a coen­zyme of E₃.

The ∆GO⁰‘ is -33.5 kJ/mole (-8.0 kcal/ mole), indicating that the reaction is highly exergonic, with the- equilibrium lying well to the right. In most animal tissues, the reaction is considered irreversible.

The Krebs cycle Reactions:

The first step of the Krebs cycle is a condensation re­action between acetyl-CoA and oxaloacetate, a prod­uct of the cycle itself. The reaction is catalyzed by cit­rate synthetase, with citrate produced and CoA released for reutilization in another round of the pre­vious reaction.

The ∆G⁰‘ is -32.2 kJ/mole (-7.7 kcal/mole). This re­action is the “pacemaker” or primary rate-limiting re­action of the cycle. The rate is controlled by the avail­ability of acetyl-CoA and oxaloacetate. Succinyl-CoA, the product of the later step in the cycle, is a competi­tive inhibitor of this reaction, because it competes with acetyl-CoA for the active site on the enzyme. The enzymatic conversion of citrate to isocitrate is a two-step process in which the intermediate, cisaconitate, remains attached to the enzyme aconitase and therefore is frequently not shown in the Krebs cy­cle. The equilibrium of this reaction is toward citrate (i.e., ∆G⁰‘ is 6.7 kJ/mole or 1.59 kcal/mole), but the iso­citrate is rapidly oxidized in the next step, thus shift­ing the direction of the reaction by removal of the product.

The oxidation of isocitrate to α-ketoglutarate is also a two-step process, with the intermediate remaining at­tached to the enzyme isocitrate dehydrogenase. The first step is an oxidation, two hydrogens being trans­ferred to NAD⁺. Actually, there are two isocitrate de­hydrogenases in the mitochondrial matrix.

One is linked to NAD⁺ and the other to NADP⁺ An NADP ⁺ -linked isocitrate dehydrogenase is also found in the cytosol. However, it appears that the NAD ⁺ – isocitrate dehydrogenase is the most active form in Krebs cycle reactions. This is an allosteric enzyme and is specifically stimulated by ADP.

When large amounts of ATP are consumed in the cell and corre­sponding quantities of ADP produced the rising ADP level acts as a positive effector for the allosteric en­zyme. Alternatively, when the level of ATP rises, the accompanying fall in the level of ADP diminishes the positive effect on the allosteric enzyme. The overall ef­fect of ADP-dependent NAD ⁺-isocitrate dehydro­genase in controlling the level of Krebs cycle activity is considered secondary, however, to the citrate synthe­tase step.

The second step of the reaction catalyzed by iso­citrate dehydrogenase is the decarboxylation of the β-carboxyl group. The ∆G⁰‘ for the entire reaction is – 20.9 kJ/mole (- 5.0 kcal/mole).

The oxidation of α-ketoglutarate to succinyl-CoA in­volves an enzyme complex called the α-ketoglutarate dehydrogenase complex. In the sequence of reactions catalyzed by this complex, CO₂ is removed by first complexing with thiamine pyrophosphate. During ad­dition of CoA, two hydrogen atoms removed from lipoic acid and coenzyme A reduce NAD⁺ to NADH + H ⁺. The ∆G⁰‘ is -33.5 kJ/mole (-8.0 kcal/ mole).

The removal of CoA from succinyl-CoA is coupled to substrate-level phosphorylation of GDP and is cata­lyzed by the enzyme succinyl-CoA synthetase.

The ∆G⁰‘ is -2.9 kJ/mole (-0.7 kcal/mole). Phosphate is attached to the enzyme-succinyl-CoA complex be­fore it is transferred to GDP. In Escherichia coli, the phosphate is transferred to ADP; in animal and most plant tissues GTP is formed and then the GTP do­nates the phosphate to ADP to form ATP.

GTP + ADP-ATP + GDP …(16-11)

Succinic dehydrogenase catalyzes the oxidation of succinate to fumarate. This is the one enzyme of the TCA cycle reactions that has been shown to be at­tached to the inner surface of the inner membrane although it is easily removed from the membrane. Suc­cinic dehydrogenase employs a flavin adenine dinucleotide (FAD) as a coenzyme, and it is this coen­zyme that accepts the two hydrogens removed from the succinate during oxidation.

succinate + FAD → fumarate + FADH₂ …(16-12)

The ∆G⁰‘ is 0.

Fumarate is converted to malate by fumarase. The reaction has a ∆G⁰‘ of -3.7 kJ/mole (-0.88 kcal/ mole).

Fumarate + HgO →malate …(16-13)

Malate is oxidized by malate dehydrogenase, which is an NAD ⁺ -containing enzyme. Although the AG⁰‘ is 29.7 kJ/mole (7.1 kcal/mole) and indicates that the iso­lated reaction

malate + NAD⁺ →oxaloacetate + NADH + H ⁺ …(16-14)

is endergonic, the products of the reaction are readily removed in vivo and the reaction is therefore driven in the direction as written. Malate dehydrogenase is found not only in the matrix of mitochondria but also in the cytosol. The oxaloacetate produced by the reac­tion can serve to initiate the cycle again by combining with acetyl-CoA to form citrate.

Summary of the TCA Cycle:

It is possible to account for all the atoms that pass through the TCA cycle. There are two’ carbons in acetyl-CoA, and during the cycle one is converted to CO₂ at the isocitrate dehydrogenase step (16-8) and the other is converted to CO₂ at the α-ketoglutarate de­hydrogenase step (16-9). Although these are not the same carbon atoms that entered the cycle, a balance is achieved—two carbons enter the cycle and two leave the cycle.

The same accounting of carbons can be made when starting with glucose.

C₆H₁₂O₆ + 6 O₂-6CO₂ + 6H₂O …(16-15)

This monosaccharide is broken down during glycolysis in the cytosol (Fig. 16-19) to form two mole­cules of pyruvate. Each pyruvate molecule loses one carbon to CO₂ as it enters the Krebs cycle at the pyru­vate dehydrogenase step (16-1 and 16-2) and another at each of the two steps described above in the TCA cycle.

An accounting can also be made for the hydrogen atoms during glycolysis and Krebs cycle reactions. One glucose (C₆ H₁₂ O₆) and 6 H₂0 contribute a total of 24 hydrogens. During glycolysis, 4 hydrogens are re­moved at the glyceraldehyde-3-phosphate dehydro­genase step to form 2 NADH+ 2 H⁺ in the cytosol and the two pyruvates from glucose each yield two more hydrogens (2 NADH + 2 H⁺) at the pyruvate dehydrogenase step (16-1 and 16-2).

Four more are transferred to NAD⁺ at the isocitrate dehydrogenase step (16-8), four are transferred to NAD⁺ at the a-ketoglutarate dehydrogenase step (16- 8), four more are transferred to FAD at the succinate dehydrogenase step (16-12), and finally another four are transferred to NAD⁺ at the malate dehydro­genase step (16-14). This accounts for all 24 hydro­gens. Although these are not the identical hydrogen atoms that were present in the original glucose and water molecules, a numerical balance is nonetheless achieved.

In a similar way, the oxygens also balance. Glucose contributes 6 oxygens and the 6 water molecules added mean that 12 oxygens enter the system. Twelve oxygen molecules are present in the 6 C0₂ molecules produced. Taken altogether, the overall equation for the respiration of glucose through the Krebs cycle re­actions is

The physiological importance of the Krebs cycle reac­tions is the production of tremendous chemical reduc­ing power in the matrix of the mitochondrion by the accumulation of NADH, H⁺, and FADH₂. These com­pounds enter the electron transport system reactions and their reducing potential drives the oxidative phos­phorylation reactions that produce ATP.