16.3 The Hub of Intermediary Metabolism in Chapter 16 The Citric Acid Cycle

16.3 The Hub of Intermediary Metabolism

The eight-step cyclic process for oxidation of simple two-carbon acetyl groups to ${CO}_{2}$ $CO Subscript 2$ may seem unnecessarily complex and not in keeping with the biological principle of maximum economy. Remember, though, that the role of the citric acid cycle is not confined to the oxidation of acetate from carbohydrates, fatty acids, or amino acids. The cycle also accepts 3-, 4-, and 5-carbon skeletons, especially from the breakdown of amino acids, at other points in the pathway. For example, when deaminated, the amino acids aspartate and glutamate become the cycle intermediates oxaloacetate and α-ketoglutarate, respectively.

The Citric Acid Cycle Serves in Both Catabolic and Anabolic Processes

In aerobic organisms, the citric acid cycle is an amphibolic pathway, one that serves in both catabolic and anabolic processes. In the cycle’s anabolic role, oxaloacetate and α-ketoglutarate can be withdrawn from the cycle to serve as precursors of aspartate and glutamate by simple transamination (Chapter 22). Through aspartate and glutamate, the carbons of oxaloacetate and α-ketoglutarate are then used to build other amino acids, as well as purine and pyrimidine nucleotides. Succinyl-CoA is a central intermediate in the synthesis of the porphyrin ring of heme groups, which serve as oxygen carriers (in hemoglobin and myoglobin) and electron carriers (in cytochromes) (see Figs. 22-25, 22-26). Finally, oxaloacetate can be converted to glucose via gluconeogenesis (see Fig. 14-16).

One biosynthetic process is not possible for animals: the conversion of acetate or acetyl-CoA to glucose. Given that the carbon atoms of acetate molecules entering the citric acid cycle appear eight steps later in oxaloacetate, it might seem that the cycle could generate oxaloacetate from acetate, then the oxaloacetate could be used to synthesize glucose by gluconeogenesis. However, there is no net conversion of acetate to oxaloacetate; for every two carbons that enter the cycle as acetate (acetyl-CoA), two leave as ${CO}_{2}$ $CO Subscript 2$ . In bacteria, plants, fungi, and protists, another reaction sequence, the glyoxylate cycle, serves as a mechanism for converting acetate to carbohydrate. The glyoxylate cycle, which shares some reactions with the citric acid cycle, converts two molecules of acetate to one of oxaloacetate in a variant of the citric acid cycle in which the two decarboxylation steps are bypassed (see Fig. 20-45). Thus, plants and many simpler organisms can synthesize glucose from fatty acids, but humans and other animals cannot.

Anaplerotic Reactions Replenish Citric Acid Cycle Intermediates

Under most circumstances, there is a dynamic steady state between reactions that siphon intermediates away from the citric acid cycle and those that supply additional carbon skeletons. When the withdrawal of cycle intermediates for use in biosynthesis lowers the concentrations of citric acid cycle intermediates enough to slow the cycle, the intermediates are replenished by anaplerotic reactions (Greek, “to refill”) (Fig. 16-15). Table 16-2 shows the most common anaplerotic reactions, all of which, in various tissues and organisms, convert either pyruvate or phosphoenolpyruvate to oxaloacetate or malate. The most important anaplerotic reaction in mammalian liver, kidney, and brown adipose tissue is the reversible carboxylation of pyruvate by ${HCO}_{3}^{-}$ $HCO Subscript 3 Superscript minus$ to form oxaloacetate, catalyzed by pyruvate carboxylase. The enzymatic addition of a carboxyl group to pyruvate requires energy, which is supplied by ATP.

A figure shows the role of the citric acid cycle in anabolism, showing how citric acid cycle intermediates can be used as precursors in biosynthetic pathways. — FIGURE 16-15 Role of the citric acid cycle in anabolism. Intermediates of the citric acid cycle are drawn off as precursors in many biosynthetic pathways. Shown in red are four anaplerotic reactions that replenish depleted cycle intermediates (see Table 16-2).

The citric acid cycle is shown as a series of clockwise blue arrows in the center of the figure. At the top of the cycle, an arrow points from oxaloacetate at the eleven o’clock position to citrate at the two o’clock position. Pyruvate is shown above with a blue arrow pointing down to acetyl-Co A from which a blue line extends down to join the arrow from oxaloacetate to citrate at the twelve o’clock position. A red arrow labeled pyruvate carboxylase points from pyruvate to oxaloacetate. An arrow points from citrate to a blue box at the upper right labeled fatty acids, sterols. This blue box is the first of a series of blue boxes that form part of a circle around the outside of the citric acid cycle. A series of two clockwise blue arrows point from citrate to Greek letter alpha-ketoglutarate at the three o’clock position. An arrow points right from Greek letter alpha-ketoglutarate to the word glutamate in a blue box in the outer circle. A blue arrow points up from glutamate to arginine beneath proline beneath glutamine, and a blue arrow points down from glutamate to purines. A clockwise blue arrow points from Greek letter alpha-ketoglutarate to succinyl-Co A at just past the six o’clock position, from which a blue arrow points down to the words porphyrins, heme in a blue box below. A series of three clockwise blue arrows point from succinyl-Co A to malate at the nine o’clock position. A red arrow labeled malic enzyme points up to malate from pyruvate below. A clockwise blue arrow points from malate to oxaloacetate at the eleven o’clock position, restarting the cycle. A blue arrow points from oxaloacetate to aspartate, asparagine at the top of a blue box to the lower right. A blue arrow points down from aspartate, asparagine to pyrimidines. A left-pointing blue arrow points left from oxaloacetate to phosphoenolpyruvate (P E P). A red arrow labeled P E P carboxylase points back from phosphoenolpyruvate to oxaloacetate. A curved red arrow over the top of these two arrows is labeled P E P carboxykinase and points from phosphoenolpyruvate to oxaloacetate. Phosphoenolpyruvate has an arrow pointing up to glucose in a blue box and an arrow pointing down to a blue box containing serine, glycine, cysteine, phenylalanine, tyrosine, and tryptophan.

TABLE 16-2 Anaplerotic Reactions
Reaction	Tissue(s)/organism(s)
	Liver, kidney
	Heart, skeletal muscle
	Higher plants, yeast, bacteria
	Widely distributed in eukaryotes and bacteria

Pyruvate carboxylase is a regulatory enzyme and is virtually inactive in the absence of acetyl-CoA, its positive allosteric modulator. Whenever acetyl-CoA, the fuel for the citric acid cycle, is present in excess, it stimulates the pyruvate carboxylase reaction to produce more oxaloacetate, enabling the cycle to use more acetyl-CoA in the citrate synthase reaction.

Biotin in Pyruvate Carboxylase Carries One-Carbon ( ${CO}_{2}$ $bold CO Subscript bold 2$ ) Groups

The pyruvate carboxylase reaction requires the vitamin biotin (Fig. 16-16), which is the prosthetic group of the enzyme. Biotin, which plays a key role in many carboxylation reactions, is a specialized carrier of one-carbon groups in their most oxidized form: ${CO}_{2}$ $CO Subscript 2$ . (The transfer of one-carbon groups in more reduced forms is mediated by other cofactors, notably tetrahydrofolate and S-adenosylmethionine, as described in Chapter 18.) Carboxyl groups are activated in a reaction that consumes ATP and joins ${CO}_{2}$ $CO Subscript 2$ to enzyme-bound biotin. This “activated” ${CO}_{2}$ $CO Subscript 2$ is then passed to an acceptor (pyruvate in this case) in a carboxylation reaction.

A figure shows the role of biotin in the reaction catalyzed by pyruvate carboxylase in seven steps. — MECHANISM FIGURE 16-16 The role of biotin in the reaction catalyzed by pyruvate carboxylase. Biotin is attached to the enzyme through an amide bond with the ε-amino group of a Lys residue, forming biotinyl-enzyme. Biotin-mediated carboxylation reactions occur in two phases, generally catalyzed in separate active sites on the enzyme, as exemplified by the pyruvate carboxylase reaction. In the first phase (steps to ), bicarbonate is converted to the more activated $CO Subscript 2$ $CO Subscript 2$ , and then used to carboxylate biotin. The biotin acts as a carrier to transport the ${CO}_{2}$ $CO Subscript 2$ from one active site to another on an adjacent monomer of the tetrameric enzyme (step ). In the second phase (steps to ), catalyzed in this second active site, the ${CO}_{2}$ $CO Subscript 2$ reacts with pyruvate to form oxaloacetate.

MECHANISM FIGURE 16-16 The role of biotin in the reaction catalyzed by pyruvate carboxylase. Biotin is attached to the enzyme through an amide bond with the ε-amino group of a Lys residue, forming biotinyl-enzyme. Biotin-mediated carboxylation reactions occur in two phases, generally catalyzed in separate active sites on the enzyme, as exemplified by the pyruvate carboxylase reaction. In the first phase (steps to ), bicarbonate is converted to the more activated $CO Subscript 2$ $CO Subscript 2$ , and then used to carboxylate biotin. The biotin acts as a carrier to transport the ${CO}_{2}$ $CO Subscript 2$ from one active site to another on an adjacent monomer of the tetrameric enzyme (step ). In the second phase (steps to ), catalyzed in this second active site, the ${CO}_{2}$ $CO Subscript 2$ reacts with pyruvate to form oxaloacetate.

Bicarbonate is shown as a red highlighted molecule with a central C double bonded to O to the upper left, bonded to O to the lower left further bonded to H below, and bonded to O minus to the right. A T P is shown to its right has a rectangle labeled adenosine bonded to a vertical chain of a circle labeled P bonded to a circle labeled P bonded to O bonded to P double bonded to O to the right and bonded to O minus below and to the left. Curved red arrows point from O minus at the right side of bicarbonate to P of the bottom phosphate group of A T P and from the bond between P of the bottom phosphate group of A T P and the O above it. To the lower right, pyruvate carboxylase is shown as a curved surface of pyruvate carboxylase with crescent-shaped pieces on the left and right ends labeled as catalytic site 1 and catalytic site 2, respectively. Biotinyl-lysine is shown extending up from L y s in the center of pyruvate carboxylase. L y s has a bond that extends into the open region above pyruvate carboxylase, where it joins N bonded to H and bonded to a five-carbon zigzag chain with the first carbon, adjacent to N H, double bonded to O, and the fifth carbon at the top bonded to the lower right vertex of a five-membered ring joined with another five-membered ring above. The bottom five-membered ring has S at its bottom vertex, to the left of the lower right vertex bonded to the vertical zigzag chain, and shares its top side with the bottom side of another five-carbon ring that has N substituted for C and bonded to H at its upper left and right vertices and its top vertex double bonded to O. Step 1: Bicarbonate is activated by A T P, forming carboxyphosphate. An arrow points right with a curved arrow that branches away to show the loss of A D P. This yields carboxyphosphate, shown in brackets as a red highlighted piece bonded to phosphate. It has red highlighted C connected by a red double bond to red highlighted O to its upper left, connected by a red bond to red highlighted O to its lower left further connected by a red bond to red highlighted H below, and a nonhighlighted bond to O further bonded to P double bonded to O above and bonded to O minus to the right and below. Curved red arrows are shown from the bond between red highlighted O and H beneath the central red highlighted C to the bond between the same red highlighted O and red highlighted C and from the bond between the red highlighted C and the nonhighlighted O to its right to the same O. Step 2: Carboxyphosphate breaks down to C O 2. A right-pointing arrow has a curved arrow branching away to show the loss of P subscript i. This yields a product of red highlighted C connected by red double bonds to red highlighted O to its upper left and lower right next to biotinyl-lysine that now has a pair of red electrons on N at the upper left vertex of the top ring. Curved red arrows point from the pair of red electrons to the red highlighted C and from the red double bond between the same C and O to its lower right and the O to it lower right. Step 3: C O 2 reacts with biotin to form carboxybiotin. An arrow points down accompanied by a curved arrow that branches away to show the loss of H plus. This yields a similar molecule except that N H at the upper left vertex of the top ring of biotin is bonded to red highlighted C to its left connected by a red double bond to O to its upper left and by a red bond to O minus to its lower left. Step 4: Biotin transports the C O 2 from one active site to the other. An arrow points down. A similar structure is shown except that biotin has changed its orientation so that the first C of the zigzag chain is now double bonded to O to the left and the N that had been at the upper left vertex of the top ring is now at the upper right vertex, where it is bonded to red highlighted C connected by a red double bond to O to its upper right and by a red bond to O minus to its lower right. Curved red arrows point from the double bond between the top vertex of the upper five-membered ring and O to the same O, from the bond between the red highlighted C and N at the upper right vertex of the top ring to the upper right bond of the same ring, and from the negative sign on the red highlighted O to the bond between the same O and the red highlighted C. Step 5: Biotin is decarboxylated. Pyruvate is converted to its enolate form. An arrow points right and is joined by a line showing the addition of pyruvate. This yields a similar structure except that the upper ring of biotin now has a double bond at the upper right side and the top vertex has a single bond to O minus above. A red highlighted molecule with C double bonded to O on each side is shown. To the right of the upper ring of biotin, a pyruvate molecule is shown with a vertical three-carbon chain with the top carbon bonded to O minus to the upper left and double bonded to O to the upper right, the middle carbon double bonded to O to the right, and the bottom carbon bonded to 2 H and to H below. Curved red arrows point from the negative charge on O bonded to the top vertex of the top ring to the bond connecting the same O to the top vertex, from the double bond at the upper right side of the top ring to H bonded beneath the bottom C of the pyruvate, from the bond between H bonded to the bottom C of pyruvate and the bond between the bottom and middle carbons of pyruvate, and from the double bond between the middle carbon of pyruvate and oxygen to the same oxygen. Step 6: Pyruvate enolate reacts with C O 2 to form oxaloacetate. An arrow points to the right. Biotin appears similar except that there is a double bond between the top vertex and O and N at the upper right vertex is bonded to H. Pyruvate enolate is shown as a vertical three-carbon chain with the top C bonded to O minus to the upper left and double bonded to O to the upper right, the middle C bonded to O minus to the right and double bonded to the bottom C below, and the bottom C bonded to 2 H. The red highlighted molecule with C double bonded to O on each side is also present. Curved red arrows point from the negative charge on O bonded to the middle C of pyruvate enolase to the bond between the same O and the middle C, from the double bond between the middle and bottom carbons of pyruvate enolate and the red highlighted C in C O 2, and from the double bond between C in C O 2 and O to its lower right and the same O. Step 7: Oxaloacetate is released. This yields oxaloacetate, shown as a vertical four-carbon chain with the top carbon bonded to O minus to the upper left and double bonded to O to the upper right, the second carbon double bonded to O to the right, the third carbon bonded to 2 H, and the red highlighted fourth carbon connected by a red double bond to red highlighted O to the lower right and connected by a red bond to red highlighted O minus at the lower right.

Pyruvate carboxylase has four identical subunits, each containing a molecule of biotin covalently attached through an amide linkage to the ε-amino group of a specific Lys residue in the enzyme active site. Carboxylation of pyruvate proceeds in two steps (Fig. 16-16): first, a carboxyl group derived from ${HCO}_{3}^{-}$ $HCO Subscript 3 Superscript minus$ is attached to biotin, then the carboxyl group is transferred to pyruvate to form oxaloacetate. These two steps occur at separate active sites; the long flexible arm of biotin transfers activated carboxyl groups from the first active site (on one monomer of the tetramer) to the second (on the adjacent monomer), functioning much like the long lipoyllysyl arm of $E_{2}$ $upper E Subscript 2$ in the PDH complex (Fig. 16-6) and the long arm of the CoA-like moiety in the acyl carrier protein involved in fatty acid synthesis (see Fig. 21-5); these are compared in Figure 16-17. Lipoate, biotin, and pantothenate all enter cells on the same transporter; all become covalently attached to proteins by similar reactions; and all provide a flexible tether that allows bound reaction intermediates to move from one active site to another in an enzyme complex, without dissociating from it. That is, all participate in substrate channeling.

A figure shows the structures of three biological tethers involving the cofactors lipoate, biotin, and the combination of Greek letter beta-mercaptoethylamine and pantothenate. — FIGURE 16-17 Biological tethers. The cofactors lipoate, biotin, and the combination of β-mercaptoethylamine and pantothenate form long, flexible arms (green) on the enzymes to which they are covalently bound, acting as tethers that move intermediates from one active site to the next. The group shaded light red is, in each case, the point of attachment of the activated intermediate to the tether.

The top molecule has a large green sphere labeled dihydrolipoyl transacetylase (E subscript 2) at the right side. Two bonds extend from its left side. The top bond extends to C double bonded to O above, from which a bond extends down to the left to C H. The bottom bond extends to N bonded to H, from which a bond extends up to the left to the same C H, forming a triangular opening between the upper and lower chains and dihydrolipoyl transacetylase. A four-carbon zigzag chain extends to the left from the C H where the right-hand chains join, then bonds to N. A bracket shows that the entire piece from this N to the bonds connecting these chains to dihydrolipoyl transacetylase represent a lysine residue. N is further bonded to a five-carbon zigzag chain with the first carbon, adjacent to N, double bonded to O above and the terminal carbon bonded to the upper right vertex of a five-membered ring that has S substituted for C at the lower left and right vertices with both S atoms and the bond connecting them highlighted in red. The part of the structure to the left of the N at the end of the L y s residue is labeled lipoate. The middle molecule has a medium-sized green sphere labeled pyruvate carboxylase at the left side. To its left, there is a L y s residue similar to the one in the top molecule and joined to a similar five-carbon chain with its first carbon, adjacent to N, double bonded to O above. The five-carbon chain ends by bonding to the lower right vertex of a five-membered ring with S at the bottom vertex and a top side shared with the bottom side of a second five-membered ring above in the opposite orientation (with a single vertex pointing up instead of down). The top five-membered ring has N substituted for C at the upper left and right vertices and each bonded to H with the left-hand N H enclosed in a red box. The top vertex is double bonded to O. The part of the molecule to the left of the terminal N of the L y s residue is labeled biotin. The bottom molecule has a small green sphere labeled acyl carrier protein at its right side. This has a bond extending to S e r to the left, from which a bond extends to O bonded to P double bonded to O above, bonded to O minus below, and bonded to O to the left that begins an eight-member pantothenate chain. Beginning at O on its right side, this chain has C H 2, C bonded to 2 C H 3 above, C bonded to O H below, C double bonded to O above, N substituted for C and bonded to H, C H 2, C H 2, and C double bonded to O above and further bonded to N outside of pantothenate. This N is bonded to H and forms the right end of Greek letter beta-mercaptoethylamine. N is bonded to a two-carbon zigzag chain that ends at S H in a red box.

SUMMARY 16.3 The Hub of Intermediary Metabolism

The citric acid cycle is amphibolic, serving in both catabolism and anabolism. Besides acetyl-CoA, any compound that gives rise to a four- or five-carbon intermediate of the citric acid cycle — for example, the breakdown products of many amino acids — can be oxidized by the cycle. Cycle intermediates can be drawn off and used as the starting material for a variety of biosynthetic products.
When intermediates are shunted from the citric acid cycle to other pathways, they are replenished by several anaplerotic reactions, which produce four-carbon intermediates by carboxylation of three-carbon compounds; pyruvate carboxylase is a major anaplerotic enzyme.
Enzymes that catalyze carboxylations commonly employ biotin to activate ${CO}_{2}$ $CO Subscript 2$ and to carry it to acceptors such as pyruvate or phosphoenolpyruvate.