Citric Acid Cycle
To date you have learned about metabolic oxidations converting glucose to acetyl-CoA. Acetyl-CoA is probably the most common metabolic intermediate found in cells. Acetyl-CoA is an "activated" two carbon compound found in many central metabolic pathways, including the citric acid cycle, the glyoxylate cycle, fatty acid synthesis, fatty acid oxidation, isoprene metabolism, amino sugar metabolism, ketone body metabolism, and cholesterol biosynthesis. The term "activated" used to describe the compound comes partly from the nature of the high energy thioester bond in the molecule with a DeltaG0' of -31.5 kJ/mol. Acetyl-CoA is one of the most ubiquitous metabolites in biological systems.
Acetyl-CoA is also an allosteric regulator of the enzymes pyruvate kinase (turns it off) and pyruvate carboxylase (turns it on). Acetyl-CoA can be produced, as you have seen, from glucose and is also produced as a breakdown product of fatty acids, some amino acids, and other metabolic compounds. Acetyl-CoA is also useful as a building block of fatty acids (indirectly), amino acids, steroids, prostaglandins, nucleotides, and other compounds. As you will see this quarter, acetyl-CoA, thus figures prominently in several major metabolic pathways.
Ubiquity of the Cycle
The citric acid cycle (named for citrate) occurs in virtually every living cell known (it is NOT unique to citrus fruit) (Figure 14.1). Specifically, in eukaryotic cells, the citric acid cycle (also called the TriCarboxylic Acid (TCA) Cycle) occurs in the matrix of the mitochondria. The cycle provides the primary means for oxidizing the acetyl carbons in acetyl-CoA to carbon dioxide. Unlike most metabolic pathways, the reactions of the citric acid cycle are written in a circle, because the end product of one cycle (oxaloacetate) becomes the starting substrate to which an acetyl group is attached in the next cycle. Students should be aware that though the pathway is written in a circle, it is NOT perfectly cyclic in that each "turn" of the cycle does not result in regeneration of all of the starting materials. Instead, a cycle consists of addition of an acetyl group from acetyl-CoA to oxaloacetate to form citrate plus CoASH. Two decarboxylation reactions occur in the cycle to yield two carbon dioxide molecules and the oxaloacetate is regenerated to begin another cycle. The net result is that one turn of the citric acid cycle yields two carbon dioxides and energy stored in electron carriers (NADH and FADH2).
Students should also note that the two carbons released as carbon dioxide in the citric acid cycle do not come from the acetyl group added at the beginning of the cycle. Rather, the carbons in carbon dioxide come from acetyl groups added in earlier cycles. Thus, it takes at least two turns of the citric acid cycle for the carbons from an acetyl-CoA to appear as carbon dioxide.
Organization of Cycle
The reactions of the citric acid cycle can be broken down in to two distinct phases. In the first part, two carbons from citrate are oxidized to CO2, yielding an activated four carbon compound (succinyl-CoA).
In the second part of the citric acid cycle, succinyl-CoA is converted back to oxaloacetate (see also Figure 14.3), thus making it possible to begin the process again. In each turn of the citric acid cycle, two carbons are added from acetyl-CoA and two carbons are oxidized as CO2. Thus, there is no net incorporation of carbons from acetyl-CoA in each turn of the cycle.
We often hear the term "respiration" used in conjunction with the citric acid cycle. Actually, respiration is much broader than just what occurs in this cycle (Figure 14.2). The term respiration is used to refer to the process in which cellular energy is generated through the oxidation of nutrient molecules, with O2 as the ultimate electron acceptor. This type of respiration is also called cellular respiration to distinguish it from the respiration of breathing. We shall talk more about repiration when we deal with the electron transport system and oxidative phosphorylation. Click here for more on respiration.
Citric Acid Cycle Reactions
The citric acid cycle is a central metabolic pathway which generates NADH and FADH2 for use in electron transport . It also produces GTP via substrate-level phosphorylation. Many metabolic processes use intermediates of the citric acid cycle in their pathways. The cyclic process is generally considered to "begin" with addition of acetyl-CoA to oxaloacetate to form citrate. Remember, however, that the pathway is cyclic in nature.
Reaction Summary of the Citric Acid Cycle (Table 14.1):
1. Acetyl-CoA + Oxaloacetate + H2O <=> Citrate + CoASH + H+ (Enzyme: <<Citrate Synthase>>, DeltaG0'= -32.2 kJ/mol)
[Note that the large negative Delta G0' value of this reaction helps to "pull" the highly positive of the reaction preceding it. Citrate synthase is regulated by availability of substrates - acetyl-CoA and oxaloacetate. Citroyl-CoA is a transient intermediate in the reaction. Citrate is used in fatty acid biosynthesis to transport acetyl-CoA across the mitochondrial membrane to the cytoplasm (Figure 18.31). Citrate acts allosterically to stimulate polymerization of acetyl-CoA carboxylase (regulatory enzyme for fatty acid biosynthesis) and inhibits the glycolysis enzyme, phosphofructokinase. See also - Figure 14.11]
2. Citrate <=> cis-Aconitate + H2O <=> Isocitrate (Enzyme: <<Aconitase>>, DeltaG0'= +6.3 kJ/mol)
[The reaction is interesting in that the starting compound, citrate, has an axis of symmetry, yet a stereospecific product, D-isocitrate, is produced. This arises from the fact that aconitase has an asymmetric binding site for citrate (see Figure 14.13). Aconitase is inhibited by fluorocitrate, which can be made by treating cells with fluoracetate, which gets converted to fluoroacetyl-CoA and then combined with oxaloacetate to form fluorocitrate.]
3. Isocitrate + NAD+ <=> Alpha-Ketoglutarate + CO2 + NADH (Enzyme: <<Isocitrate Dehydrogenase>>, DeltaG0' = -20.9 kJ/mol)
[Isocitrate dehydrogenase is an allosteric control point in the citric acid cycle. In many cells, isocitrate dehydrogenase is activated by ADP and inhibited by NADH. An NADP+ specific form of the enzyme is present in both cytosol and mitochondria.]
4. Alpha-Ketoglutarate + NAD+ + CoASH <=> Succinyl-CoA + CO2 + NADH (Enzyme: <<Alpha Ketoglutarate Dehydrogenase Complex>>, DeltaG0'= -33.5 kJ/mol)
[The enyzyme uses thiamine pyrophosphate as a coenzyme and is very similar in mechanism of action to the pyruvate dehydrogenase complex (Figure 14.10). AKGDH exists as a complex, similar to the pyruvate dehydrogenase complex, with three analogous enzyme activities and the same five coenzymes - thiamine pyrophosphate, NAD+, FAD, lipoic acid, and CoASH. Notably, the AKGDH complex differs from the pyruvate dehydrogenase complex in that the regulatory activities associated with the pyruvate dehydrogenase complex are not present in the alpha-ketoglutarate dehydrogenase complex.]
5. Succinyl-CoA + Pi + GDP <=> Succinate + GTP + CoASH (Enzyme: <<Succinyl-CoA Synthetase>>, DeltaG0'= -2.9 kJ/mol)
[Note that the enzyme is named for the reverse reaction. Plants and bacteria form ATP from ADP instead of using GDP/GTP. GTP and ATP can be interconverted in the reaction catalyzed by NDPK. This reaction contains the only substrate-level phosphorylation in the citric acid cycle. See also Figure 14.15]
6. Succinate + FAD (enzyme bound) <=> Fumarate + FADH2 (Enzyme: <<Succinate Dehydrogenase>>, DeltaG0'= 0 kJ/mol)
[In the reaction, a trans double bond is formed, with transfer of the two hydrogens to FAD, forming FADH2. FAD is bound covalently to the enzyme protein through a specific histidine residue. Succinate dehydrogenase is tightly bound to the mitochondrial inner membrane. The importance of this binding is that the reduced flavin, which must be reoxidized for the enzyme to act again, becomes reoxidized through interaction with the mitochondrial electron transport system, also bound to the membrane. Succinate dehydrogenase is inhibited by malonate.]
7. Fumarate + H2O <=> L-Malate (Enzyme: <<Fumarate Hydratase>>, DeltaG0'= -3.8 kJ/mol)
[The enzyme is stereospecific, working only on the trans isomer. The enzyme is also called fumarase]
8. L-Malate + NAD+ <=> Oxaloacetate + NADH + H+ (Enzyme: <<Malate Dehydrogenase>>, DeltaG0'= +29.7 kJ/mol)
[This highly endergonic reaction proceeds to produce oxaloacetate because the highly exergonic citrate synthase reaction keeps intramitchondrial oxaloacetate levels exceedingly low (below 1uM). Plants have one form of the enzyme in glyoxysomes and another in mitochondria.]
Regulation of the Citric Acid Cycle
The citric acid cycle can be regulated allosterically at several places (Figure 14.16), but a prime control of the cycle is the availability of NAD+. As we shall see later, the relative amounts of NAD+ and NADH are controlled by the processes of electron transport and oxidative phosphorylation, which, like the citric acid cycle, occur in the mitochondria. When oxygen concentrations are low, the electron transport system is inhibited, and NADH accumulates. This reduces the amount of NAD+ available for the citric acid cycle. Thus, the more "aerobic" one is, the more the citric acid cycle runs. The primary allosterically controlled enzymes of the citric acid cycle are isocitrate dehydrogenase (activated by ADP, inhibited by NADH, inactivated by phosphorylation) and alpha ketoglutarate dehydrogenase (inhibited by succinyl-CoA and NADH). Production of acetyl-CoA is also another limitation of the cycle. Though acetyl-CoA can come from many sources, the route catalyzed by pyruvate dehydrogenase can be inhibited allosterically by acetyl-CoA, NADH, and ATP. Conversely, pyruvate dehydrogenase can be activated allosterically by AMP. Last, the cycle is also regulated by the availability of substrate used by citrate synthase. This includes the amount of acetyl-CoA and oxaloacetate. Students should recognize the importance of the citric acid cycle for energy production (3 NADH, 1 FADH2, 1 GTP per acetyl-CoA) and that the cycle is largely regulated according to the energy needs/state of the cell. We shall see with electron transport and oxidative phosphorylation that the energy state of cells varies with work (exercise for muscle cells) and the amount of oxygen available. These, in turn, have effects on the citric acid cycle.
Stoichiometry of the Cycle
Starting from acetyl-CoA, the stoichiometry of the citric acid cycle is as follows:
Acetyl-CoA + 2H2O + 3NAD+ + FAD + GDP + Pi <=> 2 CO2 + 3 NADH + FADH2 + CoASH + GTP + 2H+
Starting from glucose (via glycolysis), the stoichiometry is as follows, through the citric acid cycle:
Glucose + 2H2O + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi <=> 6 CO2 + 10 NADH + 6 H+ + 2 FADH2 + 4 ATP
As we shall discover, the NADH and FADH2 molecules can be converted to ATP in oxidative phosphorylation. Depending on how they enter the electron transport cycle, electrons from NADH yield 3 ATP per NADH and 2 ATP per FADH2. Converted in this way, one molecule of glucose yields 38 ATPs.
Anaplerotic Reactions of the Citric Acid Cycle
It is common (but incorrect) to view the citric acid cycle as simply a cyclic pathway that is intimately involved in energy metabolism of the cell. In fact, the citric acid cycle intermediates serve both as biosynthetic precursors of numerous other metabolic compounds, including amino acids, nucleotides, heme, chlorophyll, fatty acids, and steroids (see Figure 14.18). One citric acid cycle intermediate, oxaloacetate, is also an intermediate in gluconeogenesis and citrate, oxaloacetate, and acetyl-CoA are also involved membrane transport mechanisms. Both alpha ketoglutarate and oxaloacetate are involved in transamination reactions, which are essential ways of moving nitrogen groups from one molecule to another in the cell. Thus, two important points to be aware of are that 1) citric acid cycle intermediate will rise and fall in concentration depending upon the needs of the cell and 2) the needs of the cell will influence the citric acid cycle via alterations in the pools of the intermediates of the cycle. It is essential for the cell to replenish citric cycle intermediates that are taken away for metabolic synthesis reactions. Cells have mechanisms of "refilling" the pools of citric acid cycle intermediates. These pathways are referred to as anapleurotic reactions.
Since the pathway is cyclic, deprivation of one intermediate in the cycle can be made up by supplying a different intermediate, followed by conversion to the deprived intermediate. Thus, cells need only one or two major anaplerotic reactions. The most common reaction in animals is the one catalyzed by pyruvate carboxylase, which converts pyruvate to oxaloacetate, using bicarbonate and ATP. This reaction, which is also a part of the gluconeogenesis pathway, is allosterically activated by acetyl-CoA. Since acetyl-CoA is also the molecule with which oxaloacetate reacts in the reaction catalyzed by citrate synthase, the pathway has a way of jump-starting itself when oxaloacetate concentrations fall too low.
In plants, a different enzyme, phosphoenolpyruvate (PEP) carboxylase catalyzes a similar reaction to pyruvate carboxylase, using PEP as a precursor, instead of pyruvate. Yet another way to make oxaloacetate involves a third enzyme, commonly called malic enzyme. It catalyzes reductive carboxylation of pyruvate (using bicarbonate and NADPH) to malate. Malate can easily be oxidized to oxaloacetate in the standard citric acid cycle reaction.
Other anaplerotic reactions involve transamination of glutamate or aspartic acid to either alpha ketoglutarate or oxaloacetate, respectively. In these reactions, removal of the amine group from the amino acid results in its replacement with a keto group, as shown below.
Transamination reactions in amino acid metabolism are as follows:
Transamination of pyruvate yields alanine;
Transamination of oxaloacetate yields aspartate;
Transamination of aspartate yields asparagine;
Transamination of alpha-ketoglutarate yields glutamate;
Transamination of glutamate yields glutamine
Last, the glyoxylate cycle, described next, provides another anaplerotic mechanism for plants and bacteria.
Because the standard citric acid cycle involves addition of two carbons in every cycle, followed by production of two carbon dioxide molecules, there is no net incorporation of carbons from acetyl-CoA into oxaloacetate. Consequently, acetyl-CoA cannot be converted into glucose (via oxaloacetate of glucoeneogenesis) in the standard citric acid cycle. Both plants and bacteria contain enzymes that allow them to bypass the decarboxylation reactions of the citric acid cycle, thus permitting the acetyl-CoAs to be ultimately converted to glucose in net amounts via oxaloacetate (Figure 14.20). This variant of the citric acid cycle is called the glyoxylate cycle, because the two carbon compound, glyoxylate, is an intermediate. Two enzymes not present in animals are necessary for the glyoxylate cycle to occur. They are isocitrate lyase (online link here - enzyme not to be confused with citrate lyase) and malate synthase (online link here). Isocitrate lyase catalyzes cleavage of isocitrate to succinate and glyoxylate. Malate synthase catalyzes addition of glyoxylate to acetyl-CoA to yield malate. Thus, the glyoxylate starts with one four carbon compound (oxaloacetate) and during the cycle two acetyl-CoAs are added. The reactions of the cycle ultimately yield succinate and malate. Conversion of these molecules to two oxaloacetates means that the two carbons from acetyl-CoA are ultimately made into oxaloacetate. This is, of course, anaplerotic, in nature. Figure 14.21 illustrates some of the complex metabolic relationships arising in plant cells from the glyoxylate cycle.
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Thioester Bond Energy