The oxidation of acetyl CoA in the TCA cycle and the conservation of this energy as NADH and FAD(2H) is essential for generation of ATP in almost all tissues in the body. In spite of changes in the supply of fuels, type of fuels in the blood, or rate of ATP utilization, cells maintain ATP homeostasis (a constant level of ATP). The rate of the TCA cycle, like that of all fuel oxidation pathways, is principally regulated to correspond to the rate of the electron transport chain, which is regulated by the ATP/ADP ratio and the rate of ATP utilization (see Chapter 18). The major sites of regulation are shown in Figure 17.8.
Two major messengers feed information on the rate of ATP utilization back to the TCA cycle: (a) the phosphorylation state of ATP, as refl ected in ATP and ADP
CO2 Oxaloacetate
CoA Acetyl CoA
Fuel oxidation
Isocitrate dehydrogenase Citrate
Isocitrate
NAD+
NAD+ NADH
NADH + H+ Malate
malate dehydrogenase
␣-Ketoglutarate dehydrogenase
Citrate synthase
H2O
FAD(2H) FAD Fumarate
Succinate
Succinyl CoA
CO2
CoA
␣-Ketoglutarate
CoA
GTP Pi GDP
O2 H2O
NAD+
NADH + H+ NAD+
H+ + NADH NADH
Citrate
ADP NADH Ca2+
NADH Ca2+
Electron- transport chain
E T C
H+ H+
ADP + Pi ATP
– + –
+ – + –
FIG. 17.8. Major regulatory interactions in the TCA cycle. The rate of ATP hydrolysis controls the rate of ATP synthesis, which controls the rate of NADH oxidation in the electron transport chain (ETC). All NADH and FAD(2H) produced by the cycle donate electrons to this chain (shown on the right). Thus, oxidation of acetyl CoA in the TCA cycle can go only as fast as electrons from NADH enter the ETC, which is controlled by the ATP and ADP content of the cells. The ADP and NADH concentrations feed information on the rate of oxidative phosphorylation back to the TCA cycle. Isocitrate dehydrogenase (DH), α-ketoglutarate DH, and malate DH are inhibited by increased NADH concentration. The NADH/
NAD⫹ ratio changes the concentration of oxaloacetate. Citrate is a product inhibitor of citrate synthase. ADP is an allosteric activator of isocitrate dehydrogenase. During muscular contraction, increased Ca2⫹ concentrations activate isocitrate DH and α-ketoglutarate dehydrogenase (as well as pyruvate dehydrogenase).
As Otto S. exercises, his myosin ATPase hydrolyzes ATP to provide the energy for movement of myofi - brils. The decrease of ATP and increase of ADP stimulates the electron transport chain to oxi- dize more NADH and FAD(2H). The TCA cycle is stimulated to provide more NADH and FAD(2H) to the electron transport chain. The activation of the TCA cycle occurs through a decrease of the NADH/NAD⫹ ratio, an increase of ADP concentration, and an increase of Ca2⫹. Al- though regulation of the transcription of genes for TCA cycle enzymes is too slow to respond to changes of ATP demands during exercise, the number and size of mitochondria increase during training. Thus, Otto S. is increasing his capacity for fuel oxidation as he trains.
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levels, and (b) the reduction state of NAD⫹, as refl ected in the ratio of NADH/
NAD⫹. Within the cell, even within the mitochondrion, the total adenine nucleo- tide pool (AMP, ADP, plus ATP) and the total NAD pool (NAD⫹ plus NADH) are relatively constant. Thus, an increased rate of ATP utilization results in a small decrease of ATP concentration and an increase of ADP. Likewise, increased NADH oxidation to NAD⫹ by the electron transport chain increases the rate of pathways producing NADH. Under normal physiological conditions, the TCA cycle and other oxidative pathways respond so rapidly to increased ATP demand that the ATP con- centration does not signifi cantly change.
A. Regulation of Citrate Synthase
The principles of pathway regulation are summarized in Table 17.1. In pathways subject to feedback regulation, the fi rst step of the pathway must be regulated so that precursors fl ow into alternative pathways if product is not needed. Citrate syn- thase, which is the fi rst enzyme of the TCA cycle, is a simple enzyme that has no allosteric regulators. Its rate is controlled principally by the concentration of oxaloacetate, its substrate, and by the concentration of citrate, a product inhibitor competitive with oxaloacetate (see Fig. 17.8). The malate-oxaloacetate equilibrium favors malate, so the oxaloacetate concentration is very low inside the mitochon- drion and is below the Km,app (apparent Michaelis constant; see Chapter 7) of ci- trate synthase. When the NADH/NAD⫹ ratio decreases, the ratio of oxaloacetate to malate increases. When isocitrate dehydrogenase is activated, the concentration of citrate decreases, thus relieving the product inhibition of citrate synthase. Thus, both increased oxaloacetate and decreased citrate levels regulate the response of citrate synthase to conditions established by the electron transport chain and oxida- tive phosphorylation. In the liver, the NADH/NAD⫹ ratio helps determine whether acetyl CoA enters the TCA cycle or goes into the alternative pathway for ketone body synthesis.
B. Allosteric Regulation of Isocitrate Dehydrogenase
Another generalization that can be made about regulation of metabolic pathways is that it occurs at the enzyme that catalyzes the rate-limiting (slowest) step in a pathway (see Table 17.1). Isocitrate dehydrogenase, which contains eight subunits,
Table 17.1 Generalizations on the Regulation of Metabolic Pathways 1. Regulation matches function. The type of regulation used depends on the function of the
pathway. Tissue-specifi c isozymes may allow the features of regulatory enzymes to match somewhat different functions of the pathway in different tissues.
2. Regulation of metabolic pathways occurs at rate-limiting steps, the slowest steps, in the pathway. These are reactions in which a small change of rate will affect the fl ux through the whole pathway.
3. Regulation usually occurs at the fi rst committed step of a pathway or at metabolic branch points. In human cells, most pathways are interconnected with other pathways and have regulatory enzymes for every branch point.
4. Regulatory enzymes often catalyze physiologically irreversible reactions. These are also the steps that differ in biosynthetic and degradative pathways.
5. Many pathways have “feedback” regulation, that is, the end product of the pathway controls the rate of its own synthesis. Feedback regulation may involve inhibition of an early step in the pathway (feedback inhibition) or regulation of gene transcription.
6. Human cells use compartmentation to control access of substrate and activators or inhibi- tors to different enzymes.
7. Hormonal regulation integrates responses in pathways requiring more than one tissue.
Hormones generally regulate fuel metabolism by a. Changing the phosphorylation state of enzymes
b. Changing the amount of enzyme present by changing its rate of synthesis (often induction or repression of mRNA synthesis) or degradation
c. Changing the concentration of an activator or inhibitor
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CHAPTER 17 ■ TRICARBOXYLIC ACID CYCLE 267
is considered one of the rate-limiting steps of the TCA cycle and is allosterically activated by ADP and inhibited by NADH. In the absence of ADP, the enzyme exhibits positive cooperativity; as isocitrate binds to one subunit, other subunits are converted to an active conformation. In the presence of ADP, all of the subunits are in their active conformation, and isocitrate binds more readily. Consequently, the Km,app (the S0.5) shifts to a much lower value. Thus, at the concentration of isocitrate found in the mitochondrial matrix, a small change in the concentration of ADP can produce a large change in the rate of the isocitrate dehydrogenase reaction. Small changes in the concentration of the product, NADH, and of the cosubstrate, NAD⫹, also affect the rate of the enzyme more than they would a nonallosteric enzyme.
C. Regulation of `-Ketoglutarate Dehydrogenase
The α-ketoglutarate dehydrogenase complex, although not an allosteric enzyme, is product-inhibited by NADH and succinyl CoA and may also be inhibited by GTP (see Fig. 17.8). Thus, both α-ketoglutarate dehydrogenase and isocitrate dehy- drogenase respond directly to changes in the relative levels of ADP and hence the rate at which NADH is oxidized by electron transport. Both of these enzymes are also activated by Ca⫹. In contracting heart muscle and possibly other muscle tis- sues, the release of Ca⫹ from the sarcoplasmic reticulum during muscle contraction may provide an additional activation of these enzymes when ATP is being rapidly hydrolyzed.
D. Regulation of TCA Cycle Intermediates
Regulation of the TCA cycle serves two functions: It ensures that NADH is gener- ated fast enough to maintain ATP homeostasis and it regulates the concentration of TCA cycle intermediates. For example, in the liver, a decreased rate of isocitrate dehydrogenase increases citrate concentration, which stimulates citrate effl ux to the cytosol. A number of regulatory interactions occur in the TCA cycle, in addition to those mentioned earlier, that control the levels of TCA intermediates and their fl ux into pathways that adjoin the TCA cycle.
V. PRECURSORS OF ACETYL CoA
Compounds enter the TCA cycle as acetyl CoA or as an intermediate that can be converted to malate or oxaloacetate. Compounds that enter as acetyl CoA are oxi- dized to CO2. Compounds that enter as TCA cycle intermediates replenish inter- mediates that have been used in biosynthetic pathways, such as gluconeogenesis or heme synthesis, but cannot be fully oxidized to CO2.
A. Sources of Acetyl CoA
Acetyl CoA serves as a common point of convergence for the major pathways of fuel oxidation. It is generated directly from the β-oxidation of fatty acids and deg- radation of the ketone bodies β-hydroxybutyrate and acetoacetate (Fig. 17.9). It is also formed from acetate, which can arise from the diet or from ethanol oxidation.
Glucose and other carbohydrates enter glycolysis, a pathway common to all cells, and are oxidized to pyruvate. The amino acids alanine and serine are also converted to pyruvate. Pyruvate is oxidized to acetyl CoA by the PDC. A number of amino acids, such as leucine and isoleucine, are also oxidized to acetyl CoA. Thus, the fi nal oxidation of acetyl CoA to CO2 in the TCA cycle is the last step in all the major pathways of fuel oxidation.
B. Pyruvate Dehydrogenase Complex
The PDC oxidizes pyruvate to acetyl CoA, thus linking glycolysis and the TCA cycle. In the brain, which is dependent on the oxidation of glucose to CO2 to fulfi ll its ATP needs, regulation of the PDC is a life-and-death matter.
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Defi ciencies of the PDC are among the most common inherited diseases leading to lactic acidemia and, similar to pyruvate carboxylase defi ciency, are grouped into the category of Leigh disease (subacute necrotizing encephalopathy). When PDC is defective, pyruvate will accumulate and ATP production will drop. The low ATP will stimulate glycolysis (see Chapter 19) to proceed anaerobically, and to do so, pyruvate is reduced to lac- tate. In its severe form, PDC defi ciency presents with overwhelming lactic acidosis at birth, with death in the neonatal period. In a second form of presentation, the lactic acidemia is moderate, but there is profound psychomotor retardation with increasing age. In many cases, concomitant damage to the brain stem and basal ganglia lead to death in infancy. The neuro- logical symptoms arise because the brain has a very limited ability to use fatty acids as a fuel and is, therefore, dependent on glucose metabolism for its energy supply.
The most common PDC genetic defects are in the gene for the α-subunit of E1. The E1α- gene is X-linked. Because of its importance in central nervous system metabolism, pyruvate dehydrogenase defi ciency is a problem in both males and females, even if the female is a carrier. For this reason, it is classifi ed as an X-linked dominant disorder.
1. STRUCTURE OF THE PDC
PDC belongs to the α-keto acid dehydrogenase complex family and thus shares structural and catalytic features with the α-ketoglutarate dehydrogenase complex and the branched-chain α-keto acid dehydrogenase complex (Fig. 17.10). It con- tains the same three basic types of catalytic subunits: (a) pyruvate decarboxylase subunits that bind thiamine pyrophosphate (E1), (b) transacetylase subunits that bind lipoate (E2), and (c) dihydrolipoyl dehydrogenase subunits that bind FAD (E3). Although the E1 and E2 enzymes in PDC are relatively specifi c for pyru- vate, the same dihydrolipoyl dehydrogenase participates in all of the α-keto acid dehydrogenase complexes. In addition to these three types of subunits, the PDC complex contains one additional subunit, an E3-binding protein (E3-BP). Each functional component of the PDC complex is present in multiple copies (e.g., bo- vine heart PDC has 30 subunits of E1, 60 subunits of E2, and 6 subunits each of E3
and E3-BP). The E1 enzyme is itself a tetramer of two different types of subunits, α and β.
+
CH2OH
CH3 C O
SCoA C OH H
C OH H
C H HO
C O
OH H
C H
The sugar, glucose CH3
C O CH2 C OH
O
The ketone body, acetoacetate
CH3 C O C OH
O
Pyruvate COOH
CH2 CH2 6 CH2 CH2 CH3
The fatty acid, palmitate
Ethanol The amino acid,
alanine CH2OH CH3 C H COO–
CH3 H3N
FIG. 17.9. Origin of the acetyl group from various fuels. Acetyl CoA is derived from the oxidation of fuels. The portions of fatty acids, ketone bodies, glucose, pyruvate, the amino acid alanine, and ethanol that are converted to the acetyl group of acetyl CoA are shown in boxes.
NADH + H+ CoASH
COO–
CO2 CH3 C
O
Thiamine – P P lipoate FAD Pyruvate dehydrogenase complex
Acetyl CoA SCoA CH3 C
O
~ Pyruvate NAD+
FIG. 17.10. The PDC catalyzes the oxidation of the α-keto acid pyruvate to acetyl CoA.
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CHAPTER 17 ■ TRICARBOXYLIC ACID CYCLE 269
2. REGULATION OF THE PDC
PDC activity is controlled principally through phosphorylation by pyruvate dehy- drogenase kinase, which inhibits the enzyme and dephosphorylation by pyruvate de- hydrogenase phosphatase, which activates it (Fig. 17.11). Pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase are regulatory subunits within the PDC complex and act only on the complex. PDC kinase transfers a phosphate from ATP to specifi c serine hydroxyl (ser-OH) groups on pyruvate decarboxylase (E1).
PDC phosphatase removes these phosphate groups by hydrolysis. Phosphorylation of just one serine on the PDC E1α-subunit can decrease its activity by over 99%.
PDC kinase is present in complexes as tissue-specifi c isozymes that vary in their regulatory properties.
PDC kinase is itself inhibited by ADP and pyruvate. Thus, when rapid ATP uti- lization results in an increase of ADP or when activation of glycolysis increases pyruvate levels, PDC kinase is inhibited and PDC remains in an active, nonphos- phorylated form. PDC phosphatase requires Ca2⫹ for full activity. In the heart, in- creased intramitochondrial Ca2⫹ during rapid contraction activates the phosphatase, thereby increasing the amount of active, nonphosphorylated PDC.
PDC is also regulated through inhibition by its products, acetyl CoA and NADH.
This inhibition is stronger than regular product inhibition because their binding to PDC stimulates its phosphorylation to the inactive form. The substrates of the en- zyme, CoASH and NAD⫹, antagonize this product inhibition. Thus, when an ample supply of acetyl CoA for the TCA cycle is already available from fatty acid oxida- tion, acetyl CoA and NADH build up and dramatically decrease their own further synthesis by PDC.
PDC can also be activated rapidly through a mechanism involving insulin, which plays a prominent role in adipocytes. In many tissues, insulin may, over time, slowly increase the amount of PDC present.
The rate of other fuel oxidation pathways that feed into the TCA cycle is also increased when ATP utilization increases. Insulin, other hormones, and diet control the availability of fuels for these oxidative pathways.