Having seen how acetyl-CoA is formed from pyruvate, we are ready to examine the citric acid cycle. An overview of how the cycle functions will be helpful. First we note a fundamental difference between glycolysis and the citric acid cycle. Glycolysis takes place by a linear sequence of enzyme-catalyzed steps, whereas the sequence of reactions in the citric acid cycle is cyclical. To begin a turn of the cycle (Fig. 15-7), acetyl-CoA donates its acetyl group to the four-carbon compound oxaloacetate to form the six-carbon citrate. Citrate is then transformed into isocitrate, also a six-carbon molecule, which is dehydrogenated with loss of CO2 to yield the five-carbon compound aketoglutarate. The latter undergoes loss of CO2 and ultimately yields the four-carbon compound succinate and a second molecule of CO2. Succinate is then enzymatically converted in three steps into the fourcarbon oxaloacetate, with which the cycle began; thus, oxaloacetate is ready to react with another molecule of acetyl-CoA to start a second turn. In each turn of the cycle, one acetyl group (two carbons) enters as acetyl-CoA and two molecules of CO2 leave. In each turn, one molecule of oxaloacetate is used to form citrate but-after a series of reactionsthe oxaloacetate is regenerated. Therefore no net removal of oxaloacetate occurs; one molecule of oxaloacetate can theoretically suffice to bring about oxidation of an infinite number of acetyl groups. Four of the eight steps in this process are oxidations, in which the energy of oxidation is conserved, with high efficiency, in the formation of reduced cofactors (NADH and FADH2).
After discussing the individual reactions of the citric acid cycle in detail, we will briefly consider the experiments that led to its discovery, and the evolutionary origins of the cycle.
Although the citric acid cycle is central to energy-yielding metabolism, its role is not limited to energy conservation. Four- and fivecarbon intermediates of the cycle serve as biosynthetic precursors for a wide variety of products. To replace intermediates removed for this purpose, cells employ anaplerotic (replenishing) reactions, which are briefly described.
Eugene Kennedy and Albert Lehninger showed in 1948 that in eukaryotes the entire set of reactions of the citric acid cycle takes place in the mitochondria. Isolated mitochondria were found to contain not only all the enzymes and coenzymes required for the citric acid cycle but also all the enzymes and proteins necessary for the last stage of respiration, namely, electron transfer and ATP synthesis by oxidative phosphorylation. As we shall see in later chapters, mitochondria also contain the enzymes that catalyze the oxidation of fatty acids to acetylCoA and the oxidative degradation of amino acids to acetyl-CoA or, for some amino acids, a-ketoglutarate, succinyl-CoA, or oxaloacetate. Thus in nonphotosynthetic eukaryotes the mitochondrion is the site of most energy-yielding oxidative reactions and of the synthesis of ATP coupled to those reactions. In photosynthetic eukaryotes, mitochondria are the major site ofATP production in the dark, but in daylight chloroplasts produce most of the ATP. Most prokaryotes contain the enzymes of the citric acid cycle in their cytosol, and their plasma membrane plays a role analogous to that of the inner mitochondrial membrane in ATP synthesis
The Citric Acid Cycle Has Eight Steps
In examining the eight successive reaction steps of the citric acid cycle, we will place special emphasis on the chemical transformations taking place as citrate formed from acetyl-CoA and oxaloacetate is oxidized to yield CO2 and the energy of this oxidation is conserved in the reduced coenzymes NADH and FADH2.(l) Formation of Citrate The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate to form citrate, catalyzed by citrate synthase:
In this reaction the methyl carbon of the acetyl group is joined to the carbonyl group (C-2) of oxaloacetate.
The citrate synthase from mitochondria has been crystallized and visualized by x-ray crystallography in the presence and absence of its substrates and inhibitors. Oxaloacetate, the first substrate to bind to the enzyme, induces a large conformational change, creating a binding site for the second substrate, acetyl-CoA. When citroyl-CoA forms on the enzyme surface, another conformational change brings the side chain of a crucial Asp residue into position to cleave the thioester. This induced fit of the enzyme first to its substrate and then to its intermediate decreases the likelihood of premature and unproductive cleavage of the thioester bond of acetyl-CoA.
| (2) Formation o f Isocitrate via
cis-Aconitate The enzyme aconitase (more
formally, aconitate hydratase) catalyzes the reversible
transformation of citrate to isocitrate, through the
intermediary formation of the tricarboxylic acid cis-aconitate,
which normally does not dissociate from the active site. Aconitase can promote
the reversible addition of H2O to the double bond of
enzyme-bound cis-aconitate in two different ways, one leading to citrate and the
other to isocitrate:
Although the equilibrium mixture at pH 7.4 and 25 °C contains less
than 10% isocitrate, in the cell the reaction is pulled to the right because
isocitrate is rapidly consumed in the subsequent step of the cycle, lowering its
steady-state concentration. Aconitase contains an iron-sulfur
center (Fig. 15-8), which acts both in the binding of the substrate at the
active site and in catalysis of the addition or removal of
H2O.
There are two different forms of isocitrate dehydrogenase (isozymes; see Box 14-3), one requiring NAD+ as electron acceptor and the other requiring NADP+ . The overall reactions catalyzed by the two isozymes are otherwise identical. The NAD-dependent enzyme is found in the mitochondrial matrix and serves in the citric acid cycle to produce a-ketoglutarate. The NADP-dependent isozyme is found in both the mitochondrial matrix and the cytosol. It may function primarily in the generation of NADPH, which is essential for reductive anabolic reactions. (4) Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2 The next step is another oxidative decarboxylation, in which a-ketoglutarate is converted to succinyl-CoA and O2 by the action of the a-ketoglutarate dehydrogenase complex; NAD+ serves as electron acceptor: This reaction is virtually identical to the pyruvate dehydrogenase reaction discussed above; both involve the oxidation of an a-keto acid with loss of the carboxyl group as CO2. The energy of oxidation of a-ketoglutarate is conserved in the formation of the thioester bond of succinyl-CoA. In both structure and function the a-ketoglutarate dehydrogenase complex closely resembles the pyruvate dehydrogenase complex. It includes three enzymes, analogous to E1, E2, and E3 of the pyruvate dehydrogenase complex, as well as enzyme-bound TPP, bound lipoate, FAD, NAD, and coenzyme A. Although El of the aketoglutarate dehydrogenase complex is structurally similar to El of pyruvate dehydrogenase, their amino acid sequences are different; the El components specifically bind either a-ketoglutarate or pyruvate, conferring substrate specificity upon their respective enzyme complexes. The subunits of E3 for the two complexes are virtually identical. The E2 components of the two complexes are very similar; both have covalently bound lipoyl moieties. It is a near certainty that the proteins of these two multienzyme complexes share a common evolutionary origin. (5) Conversion of Succinyl-CoA to Succinate Succinyl-CoA, like acetyl-CoA, has a strongly negative free energy of hydrolysis of its thioester bond (ΔG°' = -36 kJ/mol). In the next step of the citric acid cycle, energy released in the breakage of this bond is used to drive the synthesis of a phosphoanhydride bond in either GTP or ATP, and succinate is also formed in the process: The enzyme that catalyzes this reversible reaction is called either succinyl-CoA synthetase or succinic thiokinase; both names indicate the participation of a nucleoside triphosphate in the reaction . It was long thought that the enzyme from animal tissues used GDP exclusively, whereas that from plants and bacteria used predominantly ADP. It now appears that some animal cells contain two isozymes, one specific for ADP and the other for GDP. This energy-conserving reaction involves an intermediate step in which the enzyme molecule itself becomes phosphorylated at a His residue in the active site (Fig. 15-9). This phosphate group, which has a high group transfer potential, is transferred to ADP (or GDP) to form ATP (or GTP). The coupled formation of ATP (or GTP) at the expense of the energy released by the oxidative decarboxylation of α-ketoglutarate is another example of a substrate-level phosphorylation, like the synthesis of ATP coupled to the oxidation of glyceraldehyde- 3- phosphate in glycolysis (p. 411). These reactions are called substrate-level phosphorylations to distinguish them from oxidative or respirationlinked phosphorylation. Substrate-level phosphorylations involve soluble enzymes and chemical intermediates such as the phosphohistidine residue in succinyl-CoA synthetase, or 1,3-bisphosphoglycerate in the glycolytic pathway. Respiration-linked phosphorylation, on the other hand, involves membrane-bound enzymes and transmembrane gradients of protons (Chapter 18). The GTP formed by succinyl-CoA synthetase may donate its terminal phosphate group to ADP to form ATP, by the reversible action of nucleoside diphosphate kinase: Oxidation of Succinate to Fumarate The succinate formed from succinyl-CoA is oxidized to fumarate by the flavoprotein succinate dehydrogenase (right). In eukaryotes, succinate dehydrogenase is tightly bound to the inner mitochondrial membrane (in prokaryotes, to the plasma membrane); it is the only enzyme of the citric acid cycle that is membrane-bound. The enzyme from beef heart mitochondria contains three different iron-sulfur clusters as well as one molecule of covalently bound FAD. Electrons pass from succinate through the FAD and iron-sulfur centers before entering the chain of electron carriers in the mitochondrial inner membrane (or the plasma membrane of bacteria). Electron flow from succinate through these carriers to the final electron acceptor, O2, is coupled to the synthesis of two ATP molecules per pair of electrons (respiration-linked phosphorylation; Chapter 18). Malonate, an analog of succinate, is a strong competitive inhibitor of succinate dehydrogenase and therefore blocks the citric acid cycle. (7) Hydration of Fumarate to Produce Malate The reversible hydration of fumarate to L-malate is catalyzed by fumarase (fumarate hydratase):
(8) Oxidation of Malate to Oxaloacetate In the last reaction of the citric acid cycle, NAD-linked L-malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate: The equilibrium of this reaction lies far to the left under standard thermodynamic conditions. However, in intact cells oxaloacetate is continually removed by the highly exergonic citrate synthase reaction (p. 454). This keeps the concentration of oxaloacetate in the cell extremely low (< 10-6 M), pulling the malate dehydrogenase reaction toward oxaloacetate formation. |
