B. TCA Cycle as a Cyclic Pathway
TCA cycle consists of eight sequential reactions. It is begins with condensation of a four carbon oxaloacetate (OAA) molecule with an acetyl CoA molecule (2-carbon) to form a six carbon citrate molecule (Reaction 1; Fig.
9.15). In subsequent reactions of TCA cycle, two carbon atoms are lost from the citrate in the form of CO2 (Reactions 3 and 4, Fig. 9.14). A series of modifi cations occur, in the remaining four carbon atoms, in successive steps, to ultimately form oxaloacetate (OAA). Thus, the last intermediate of one cycle (i.e. OAA) is ready for use (b) to break down some compounds for generation of
energy. The energy obtained is captured by reducing NAD and FAD to NADH and FADH2, respectively.
To obtain full usage of the energy generated in this cycle, NADH and FADH2, are processed by another pathway (oxidative phosphorylation) where their energy is converted to ATP.
A. Pyruvate Dehydrogenase Complex:
A Bridge Between Glycolysis and TCA Cycle
Acetyl CoA, one of the two major reactants in the fi rst reac-tion of TCA cycle (the other being oxaloacetate), is mainly generated from pyruvate. This conversion involves a series of complex reactions, catalyzed by a multienzyme complex, called pyruvate dehydrogenase complex (PDH).
Strictly speaking, this reaction sequence is not a part of the TCA cycle. It is, however, discussed here along with TCA cycle, because it serves as a bridge between glycolysis and TCA cycle.
The pyruvate dehydrogenase complex is located in the mitochondrial matrix. It is a multimolecular aggregate (MW 9 106) consisting of three enzymes and fi ve coenzymes.
Enzymes: Pyruvate decarboxylase, dihydrolipoyl transacety-lase and dihydrolipoyl dehydrogenase are the three enzymes present in the complex. There are about 60 molecules of dihydrolipoyltransacetylase and about 20–30 molecules each of the other two enzymes in each complex.
Fig. 9.13. TCA cycle as the meeting point of various catabolic pathways.
Lipids
Fatty acids Glycerol
Carbohydrates Proteins
Amino acids Ketogenic Glucogenic
Pyruvate
Acetyl CoA
TCA
Oxaloacetate Citrate Isocitrate α-Ketoglutarate
mitochondrial membrane (Chapter 14). This arrange-ment ensures that the free energy liberated during reactions of the TCA cycle is promptly trapped in the form of ATP.
C. Reactions of TCA Cycle
The reactions brought about by different enzymes of the TCA cycle are depicted in Figure 9.16. All the enzymes are present in mitochondrial matrix except succinate dehydrogenase which is located in the inner mitochon-drial membrane.
Reaction 1: Synthesis of Citrate from Acetyl CoA and Oxaloacetate
Citrate is produced by condensation of acetyl CoA with oxaloacetate in an irreverssible reaction catalyzed by the enzyme, citrate synthase. The reaction equilibrium lies far towards the right (i.e. towards the formation of citrate).
as a substrate in the next cycle. In this way, there is no net generation of OAA, or of any of the cycle intermediates.
The reactions of TCA cycle take place in the mitochon-drial matrix. This is in close proximity to the reactions of oxidative phosphorylation, which occur in the inner
2
Fig. 9.14. Mechanism of conversion of pyruvate to acetyl CoA by pyruvate dehydrogenase complex. Step 1: Pyruvate and thia-mine pyrophosphate (TPP) combine to form a condensation product. Step 2: Pyruvate decarboxylase catalyzes release of carbon dioxide from the condensation product to form a hydroxyethyl intermediate. The latter is attached to the reactive carbon of the TPP. Step 3: Transfer of acetyl group from the hydroxyethyl intermediate to the lipoic acid (oxidized) occurs to form acetyl lipoic acid. The reaction is catalyzed by dihydrolipoyl transacetylase. Step 4: Transfer of an acetyl group from the acetyl lipoic acid to coenzyme A (CoASH) occurs next, converting the latter to acetyl CoA. The acetyl CoA then enters the TCA cycle. The other prod-uct of this reaction is reduced lipoic acid. Step 5: This step regenerates the oxidized lipoic acid (from the reduced lipoic acid), which then participates in the next cycle of reactions. This conversion is catalyzed by the dihydrolipoyl dehydrogenase component of the enzyme complex, which catalyzes transfer of the reducing equivalents fi rst to FAD and then to NAD. Overall: Pyruvate NAD CoASH Acetyl CoA CO2 NADH H.
Fig. 9.15. TCA cycle: Oxaloacetate (OAA), the last interme-diate is also a reactant in the fi rst step. The eight reactions of the pathway are numbered 1 to 8.
Acetyl CoA
Fig. 9.16. Reactions of tricarboxylic acid (Krebs) cycle.
synthetase Mg2+
Succinate
TCA cycle derives its other name (citric acid cycle) from this fi rst intermediate, citrate. However, citrate may leave the citric acid cycle to participate in other metabolic pathways as well (Fig. 9.17). Excessive citrate crosses the inner mitochondrial membrane through specifi c tricar-boxylate carriers and reaches the cytosol, where it provides acetyl CoA:
Citrate Citrate lyase
Acetyl CoA + OAA
In cytosol, acetyl CoA serves as a precursor for fatty acid synthesis (lipogenesis). Moreover, citrate stimulates
acetyl CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis. Thus citrate enhances fatty acid synthesis by:
Providing the substrate (i.e. acetyl CoA).
Stimulating the key lipogenic enzyme (i.e. acetyl CoA carboxylase).
In the cytosol, citrate has another important regula-tory role to play. It adjusts the rate of glycolysis to that of TCA cycle. It does so by inhibiting phosphofructokinase (PFK1) the rate-limiting enzyme of glycolysis (Table 9.3).
Inhibition of this enzyme decreases the rate of glycolysis,
The mechanism of this conversion is similar to that of the conversion of pyruvate to acetyl CoA (Fig. 9.14). The same set of coenzymes, i.e. thiamine pyrophosphate, lipoic acid, FAD, NAD, and CoASH, is used. Each of these performs a function analogous to that performed in the pyruvate dehydrogenase complex.
The reaction releases the second CO2 molecule of the cycle and produces the second NADH. The equilibrium of the reaction lies towards the right, i.e. towards succinyl CoA formation.
Reaction 5: Cleavage of Succinyl CoA
The enzyme succinyl CoA synthetase (also called succinate thiokinase) cleaves the high-energy thioester bond in suc-cinyl CoA (G0‘ is –8.0 kcal/mole) to release large amount of free energy, which is used to produce a GTP molecule.
This reaction provides an example of substrate level phosphorylation, since the production of a high-energy phosphate (e.g. GTP) is coupled with enzymatic transfor-mation of a substrate molecule. GTP can produce ATP by action of the enzyme nucleoside diphosphokinase.
GTP ADP GDP ATP
Note: Succinyl CoA may also serve as a substrate for haem synthesis.
Reaction 6: Oxidation of Succinate
A pair of reducing equivalents is removed from succinate by the enzyme succinate dehydrogenase to form fumarate.
FAD serves as a coenzyme in this reaction. Unlike the other enzymes of the TCA cycle, which are located in the mitochondrial matrix, succinate dehydrogenase is anchored in the inner mitochondrial membrane. It catalyzes removal of a hydrogen pair from succinate and transfers it to FAD which becomes FADH2. This in turn transfers its electrons to ubiquinone, which becomes ubiquinol, and is then transferred to complex III for oxidation (Chapter 14).
Reaction 7: Hydration of Fumarate
Addition of a water molecule to fumarate forms malate.
It is a reversible reaction, catalyzed by the enzyme fumarase.
Note: In spite of being reversible, this reaction always proceeds unidirectionally, i.e. towards formation of malate.
This is because of the thermodynamic pull in TCA cycle (discussed later).
Reaction 8: Oxidation of Malate
Removal of a pair of reducing equivalents from malate by the enzyme malate dehydrogenase produces oxaloace-tate. The reaction generates the third NADH molecule of the cycle. Malate to oxaloacetate conversion is the last reaction of the cycle. Oxaloacetate, generated in this step, and therefore, the production of acetyl CoA falls. This
results in decreased rate of TCA cycle since acetyl CoA is needed in the fi rst reaction of TCA cycle. In this way, rates of glycolysis and TCA keep pace with each other.
Thus, when citrate concentration is high, implying that the cell is adequately supplied with fuel molecules and, therefore, further production of energy is not required, the energy-yielding catabolic pathways (e.g. glycolysis and TCA cycle) are inhibited. The biosynthetic pathway (i.e.
fatty acid synthesis) is favoured at the same time. This illustrates a fundamental principle of biochemistry, i.e. the catabolic and the anabolic pathways are regulated recipro-cally. If one pathway is favoured, the other is inhibited.
Reaction 2: Isomerization of Citrate
Isomerization of citrate by the enzyme aconitase yields isocitrate. During this reaction, a transient enzyme-bound intermediate, cis-aconitate is formed.
Reaction 3: Oxidative Decarboxylation of Isocitrate The enzyme isocitrate dehydrogenase (IDH) catalyzes removal of two hydrogen atoms from isocitrate (i.e. oxi-dation) with a concomitant release of a CO2 molecule (i.e. decarboxylation). -Ketoglutarate is the reaction product.
NAD serves as a coenzyme in this step, and is con-verted to NADH by accepting a pair of hydrogen atoms.
NADP can also serve as a coenzyme in this reaction.
Isocitrate dehydrogenase is one of the few enzymes that are capable of using both NAD and NADP as coenzymes.
Reaction 4: Oxidative Decarboxylation of -Ketoglutarate Like the previous step, this one also involves oxidation and decarboxylation. The reaction is catalyzed by the enzyme -ketoglutarate dehydrogenase, to produce succi-nyl CoA.
Fig. 9.17. Citrate, a TCA intermediate, plays an important regulatory role in lipogenesis and glycolysis by modulation of activities of the enzymes, phosphofructokinase (PFK1) and acetyl CoA carboxylase (E) (IMM inner mitochondrial membrane).
Fatty acid
NADH in this manner generates three ATPs, whereas two ATPs are produced from one FADH2 molecule. In addition, one GTP is produced during enzymatic transformation of succinyl CoA (Reaction 5), which generates an ATP through action of nucleoside diphosphokinase. Hence, each cycle of TCA cycle, produces 12 ATP molecules.
3 NADH (Reaction 3,4 and 8) 9 ATP 1 FADH2 (Reaction 6) 2 ATP 1 GTP (Reaction 5) 1 ATP TOTAL (Each cycle) 12 ATP However, two acetyl CoA molecules are generated from each glucose, therefore, this cycle occurs twice, generating 6NADH, 2FADH2 and 2GTPs (Fig. 9.18) Hence , total 24 ATPs are generated. Thus, complete oxidation of glucose via glycolysis, pyruvate dehydrogenase, the Krebs cycle and the oxidative phosphorylation pathway yields 38 ATPs. Since two ATPs were initially used during stage I reactions of gly-colysis, the net yield is 36 ATPs. Compared with anaerobic pathway, the oxidative pathway thus yields 18 times more energy in the form of ATP. However, this is still far less than the energy obtained by burning of a molecule of glucose in calorimeter (2780 kJ), which is suffi cient to generate about condenses with another molecule of acetyl CoA to
initi-ate another cycle.
The following equation summarizes all the reactions of the TCA cycle.
Acetyl CoA 3NAD FAD GDP Pi 2H2O 2CO2 3NADH 2H FADH2 GTP CoA-SH Several chemicals can inhibit reactions of the TCA cycle, as outlined in Box 9.5.
A four-carbon compound oxaloacetate reacts with acetyl CoA to form six-carbon citrate, which is then converted back to oxaloacetate in the remaining reactions of the TCA cycle.
The following points about TCA cycle are noteworthy:
1. Two carbon atoms enter the cycle as acetyl CoA (and condense with oxaloacetate) and two carbons leave in the form of two molecules of CO2. Thus, TCA basically involves oxidation of acetyl CoA to carbon dioxide and, as such there is no net consumption or regeneration of oxaloacetate or any of the other cycle intermediates.
2. Some of the reactions of TCA cycle are reversible and yet they always proceed unidirectionally. This is because equilibrium of some of the reactions of TCA cycle (e.g. Reactions 1, 3 and 4, Fig. 9.12) lies far towards the right. These reactions are irreversible and have strong tendency to proceed unidirectionally, towards the right. This generates a strong “thermodynamic pull“ so that rest of the (reversible) reactions are also pulled in that direction. As mentioned earlier, fumarate to malate conversion which is freely reversible other-wise, always proceeds in direction of malate forma-tion because of the thermodynamic pull.
D. Energy Yield from TCA Cycle
There are four dehydrogenation reactions in TCA cycle (Reactions 3, 4, 6 and 8), which generate three NADH and one FADH2 molecules. These reduced coenzymes donate electrons to the electron transport chain and generate ATPs by oxidative phosphorylation. Oxidation of each
Fig. 9.18. Energetics of glucose oxidation. 38 ATPs are gen-erated by complete oxidation of glucose via glycolysis, PDH reaction, and TCA cycle.
Glucose
2 Pyruvate Pyruvate
dehydrogenase
2 NADH
2 Acetyl CoA
6 NADH 2 FADH2 2 GTP
4 CO2
TCA cycle 18 ATP
4 ATP 2 ATP 38 ATP
2 NADH 6 ATP
2 ATP
6 ATP
Glycolysis