Basic principles of metabolism: catabolism, anabolism. Common pathways of proteins, carbohydrates and lipids transformation. Studying of Krebs cycle functioning. Bioenergetics processes: biological oxidation, oxidative phosphorylation, ATP synthesis.
The fate of dietary components after digestion and absorption constitutes metabolism—the metabolic pathways taken by individual molecules, their interrelationships, and the mechanisms that regulate the flow of metabolites through the pathways. Metabolic pathways fall into three categories: (1) Anabolic pathways are those involved in the synthesis of compounds. Protein synthesis is such a pathway, as is the synthesis of fuel reserves of triacylglycerol and glycogen. Anabolic pathways are endergonic. (2) Catabolic pathways are involved in the breakdown of larger molecules, commonly involving oxidative reactions; they are exergonic,
producing reducing equivalents and, mainly via the respiratory chain, ATP. (3) Amphibolic pathways occur at the “crossroads” of metabolism, acting as links between the anabolic and catabolic pathways, eg, the citric acid cycle.
A knowledge of normal metabolism is essential for an understanding of abnormalities underlying disease. Normal metabolism includes adaptation to periods of starvation, exercise, pregnancy, and lactation. Abnormal metabolism may result from nutritional deficiency, enzyme deficiency, abnormal secretion of hormones, or
the actions of drugs and toxins. An important example of a metabolic disease is diabetes mellitus.
PATHWAYS THAT PROCESS THE MAJOR PRODUCTS OF DIGESTION
The nature of the diet sets the basic pattern of metabolism. There is a need to process the products of digestion of dietary carbohydrate, lipid, and protein. These are mainly glucose, fatty acids and glycerol, and amino acids, respectively. In ruminants (and to a lesser extent in other herbivores), dietary cellulose is fermented by symbiotic microorganisms to short-chain fatty acids (acetic, propionic, butyric), and metabolism in these animals is adapted to use these fatty acids as major substrates.
All the products of digestion are metabolized to a common product, acetyl-CoA, which is then oxidized by the citric acid cycle .
Carbohydrate Metabolism Is Centered on the Provision & Fate of Glucose
Glucose is metabolized to pyruvate by the pathway of glycolysis, which can occur anaerobically (in the absence of oxygen), when the end product is lactate. Aerobic
tissues metabolize pyruvate to acetyl-CoA, which can enter the citric acid cycle for complete oxidation to CO2 and H2O, linked to the formation of ATP.
Glucose and its metabolites also take part in other processes. Examples: (1) Conversion to the storage polymer glycogen in skeletal muscle and liver. (2) The
pentose phosphate pathway, an alternative to part of the pathway of glycolysis, is a source of reducing equivalents (NADPH) for biosynthesis and the source of ribose
for nucleotide and nucleic acid synthesis. (3) Triose phosphate gives rise to the glycerol moiety of triacylglycerols. (4) Pyruvate and intermediates of the citric acid cycle provide the carbon skeletons for the synthesis of amino acids; and acetyl-CoA, the precursor of fatty acids and cholesterol (and hence of all steroids synthesized in the body). Gluconeogenesis is the process of forming glucose from noncarbohydrate
precursors, eg, lactate, amino acids, and glycerol.
Lipid Metabolism Is Concerned Mainly With Fatty Acids & Cholesterol
The source of long-chain fatty acids is either dietary lipid or de novo synthesis from acetyl-CoA derived from carbohydrate. Fatty acids may be oxidized to acetyl- CoA (β-oxidation) or esterified with glycerol, forming triacylglycerol (fat) as the body’s main fuel reserve. Acetyl-CoA formed by β-oxidation may undergo several fates:
(1) As with acetyl-CoA arising from glycolysis, it is oxidized to CO2 + H2O via the citric acid cycle.
(2) It is the precursor for synthesis of cholesterol and other steroids.
(3) In the liver, it forms ketone bodies (acetone, acetoacetate, and 3 hydroxybutyrate) that are important fuels in prolonged starvation.
Much of Amino Acid Metabolism Involves Transamination
The amino acids are required for protein synthesis. Some must be supplied in the diet (the essential amino acids) since they cannot be synthesized in the body. The remainder are nonessential amino acids that are supplied in the diet but can be formed from metabolic intermediates by transamination, using the amino nitrogen
from other amino acids. After deamination, amino nitrogen is excreted as urea, and the carbon skeletons that remain after transamination (1) are oxidized to CO2 via the citric acid cycle, (2) form glucose (gluconeogenesis), or (3) form ketone bodies.
Several amino acids are also the precursors of other compounds, eg, purines, pyrimidines, hormones such as epinephrine and thyroxine, and neurotransmitters.
METABOLIC PATHWAYS MAY BE STUDIED AT DIFFERENT LEVELS
In addition to studies in the whole organism, the location and integration of metabolic pathways is revealed by studies at several levels of organization. At the tissue
and organ level, the nature of the substrates entering and metabolites leaving tissues and organs is defined. At the subcellular level, each cell organelle (eg, the mitochondrion) or compartment (eg, the cytosol) has specific roles that form part of a subcellular pattern of metabolic pathways.
At the Tissue and Organ Level, the Blood Circulation Integrates Metabolism
Amino acids resulting from the digestion of dietary protein and glucose resulting from the digestion of carbohydrate are absorbed and directed to the liver via the
hepatic portal vein. The liver has the role of regulating the blood concentration of most water-soluble metabolites In the case of glucose, this is achieved by taking up glucose in excess of immediate requirements and converting it to glycogen.
Between meals, the liver acts to maintain the blood glucose concentration from
glycogen (glycogenolysis) and, together with the kidney, by converting noncarbohydrate metabolites such as lactate, glycerol, and amino acids to glucose (gluconeogenesis). Maintenance of an adequate concentration of blood glucose is vital for those tissues in which it is the major fuel (the brain) or the only fuel (the erythrocytes).
The liver also synthesizes the major plasma proteins (eg, albumin) and deaminates amino acids that are in excess of requirements, forming urea, which
is transported to the kidney and excreted. Skeletal muscle utilizes glucose as a fuel, forming both lactate and CO2. It stores glycogen as a fuel for its use in muscular contraction and synthesizes muscle protein from plasma amino acids. Muscle accounts for approximately 50% of body mass and consequently represents a considerable store of protein that can be drawn upon to supply amino acids for gluconeogenesis in starvation.
Lipids in the diet are mainly triacylglycerol and are hydrolyzed to monoacylglycerols and fatty acids in the gut, then reesterified in the intestinal mucosa. Here they are packaged with protein and secreted into the lymphatic system and thence into the
blood stream as chylomicrons, the largest of the plasma lipoproteins. Chylomicrons also contain other lipidsoluble nutrients, eg, vitamins. Unlike glucose and amino acids, chylomicron triacylglycerol is not taken up directly by the liver. It is first metabolized by tissues that have lipoprotein lipase, which hydrolyzes the triacylglycerol, releasing fatty acids that are incorporated into tissue lipids or oxidized as fuel. The other major source of long-chain fatty acid is synthesis (lipogenesis) from carbohydrate, mainly in adipose tissue and the liver. Adipose tissue triacylglycerol is the main fuel reserve of the body. On hydrolysis (lipolysis) free fatty acids are released into the circulation. These are taken up by most
tissues (but not brain or erythrocytes) and esterified to acylglycerols or oxidized as a fuel. In the liver, triacylglycerol arising from lipogenesis, free fatty acids, and
chylomicron remnants is secreted into the circulation as very low density lipoprotein (VLDL). This triacylglycerol undergoes a fate similar to that of chylomicrons. Partial oxidation of fatty acids in the liver leads to ketone body production Ketone bodies are transported to extrahepatictissues, where they act as a fuel source in starvation.
At the Subcellular Level, Glycolysis Occurs in the Cytosol & the Citric Acid Cycle in the Mitochondria
Compartmentation of pathways in separate subcellular compartments or organelles permits integration and regulation of metabolism. Not all pathways are of equal
importance in all cells. Depicts the subcellular compartmentation of metabolic pathways in a hepatic parenchymal cell.
The central role of the mitochondrion is immediately apparent, since it acts as the focus of carbohydrate, lipid, and amino acid metabolism. It contains the enzymes
of the citric acid cycle, â-oxidation of fatty acids, and ketogenesis, as well as the respiratory chain and ATP synthase. Glycolysis, the pentose phosphate pathway, and fatty acid synthesis are all found in the cytosol. In gluconeogenesis, substrates such as lactate and pyruvate, which are formed in the cytosol, enter the mitochondrion to
yield oxaloacetate before formation of glucose. The membranes of the endoplasmic reticulum contain the enzyme system for acylglycerol synthesis, and the ribosomes are responsible for protein synthesis.
• The products of digestion provide the tissues with the building blocks for the biosynthesis of complex molecules and also with the fuel to power the living
• Nearly all products of digestion of carbohydrate, fat, and protein are metabolized to a common metabolite, acetyl-CoA, before final oxidation to CO2 in the
citric acid cycle.
• Acetyl-CoA is also used as the precursor for biosynthesis of long-chain fatty acids; steroids, including cholesterol; and ketone bodies.
• Glucose provides carbon skeletons for the glycerol moiety of fat and of several nonessential amino acids.
• Water-soluble products of digestion are transported directly to the liver via the hepatic portal vein. The liver regulates the blood concentrations of glucose
and amino acids.
• Pathways are compartmentalized within the cell. Glycolysis, glycogenesis, glycogenolysis, the pentose phosphate pathway, and lipogenesis occur in the cytosol.
The mitochondrion contains the enzymes of the citric acid cycle, β-oxidation of fatty acids, and of oxidative phosphorylation. The endoplasmic reticulum also contains the enzymes for many other processes, including protein synthesis, glycerolipid
formation, and drug metabolism.
• Metabolic pathways are regulated by rapid mechanisms affecting the activity of existing enzymes, eg, allosteric and covalent modification (often in response
to hormone action); and slow mechanisms affecting the synthesis of enzymes.
BIOMEDICAL IMPORTANCE OF KREBS CYCLE
The citric acid cycle ( tricarboxylic acid cycle) is a series of reactions in mitochondria that oxidize acetyl residues (as acetyl-CoA) and reduce coenzymes that upon reoxidation are linked to the formation of ATP.
The citric acid cycle is the final common pathway for the aerobic oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino
acids are metabolized to acetyl-CoA or intermediates of the cycle. It also has a central role in gluconeogenesis, lipogenesis, and interconversion of amino acids. Many
of these processes occur in most tissues, but the liver is the only tissue in which all occur to a significant extent.
The repercussions are therefore profound when, for example, large numbers of hepatic cells are damaged as in acute hepatitis or replaced by connective tissue (as in
cirrhosis). Very few, if any, genetic abnormalities of citric acid cycle enzymes have been reported; such abnormalities would be incompatible with life or normal
THE CITRIC ACID CYCLE PROVIDES SUBSTRATE FOR THE
The cycle starts with reaction between the acetyl moiety of acetyl-CoA and the four-carbon dicarboxylic acid oxaloacetate, forming a six-carbon tricarboxylic acid, citrate. In the subsequent reactions, two molecules of CO2 are released and oxaloacetate is regenerated.
Only a small quantity of oxaloacetate is needed for the oxidation of a large quantity of acetyl-CoA; oxaloacetate
may be considered to play a catalytic role. The citric acid cycle is an integral part of the process by which much of the free energy liberated during the oxidation of fuels is made available. During oxidation of acetyl-CoA, coenzymes are reduced and subsequently reoxidized in the respiratory chain, linked to the formation
of ATP. This process is aerobic, requiring oxygen as the final oxidant of the reduced coenzymes. The enzymes of the citric acid cycle are located in the mitochondrial matrix, either free or attached to the inner mitochondrial membrane, where the enzymes of the respiratory chain are also found.
REACTIONS OF THE CITRIC ACID CYCLE LIBERATE REDUCING
EQUIVALENTS & CO2
The initial reaction between acetyl-CoA and oxaloacetate to form citrate is catalyzed by citrate synthase which forms a carbon-carbon bond between the methyl carbon of acetyl-CoA and the carbonyl carbon of oxaloacetate.
The thioester bond of the resultant citryl-CoA is hydrolyzed, releasing citrate and CoASH—an exergonic reaction. Citrate is isomerized to isocitrate by the enzyme
aconitase (aconitate hydratase); the reaction occurs in two steps: dehydration to cis-aconitate, some of which remains bound to the enzyme; and rehydration to isocitrate.
Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of
the cycle are not those that were added from acetyl- CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto
the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source
of acetyl-CoA for fatty acid synthesis. The poison fluoroacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits
aconitase, causing citrate to accumulate. Isocitrate undergoes dehydrogenation catalyzed by isocitrate dehydrogenase to form, initially, oxalosuccinate,
which remains enzyme-bound and undergoes decarboxylation to α-ketoglutarate. The decarboxylation requires Mg2+ or Mn2+ ions. There are three isoenzymes of isocitrate dehydrogenase. One, which uses NAD+, is found only in mitochondria. The other two use NADP+ and are found in mitochondria and the cytosol. Respiratory
chain-linked oxidation of isocitrate proceeds almost completely through the NAD+-dependent enzyme. α-Ketoglutarate undergoes oxidative decarboxylation
in a reaction catalyzed by a multi-enzyme complex similar to that involved in the oxidative decarboxylation of pyruvate. The ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate dehydrogenase complex—thiamin diphosphate, lipoate, NAD+, FAD, and CoA—and results in the formation of succinyl-CoA. The equilibrium of this reaction is so much in favor of succinyl-CoA formation that it must be considered physiologically unidirectional.
As in the case of pyruvate oxidation, arsenite inhibits the reaction, causing the substrate ketoglutarate, to accumulate. Succinyl-CoA is converted to succinate by the enzyme succinate thiokinase (succinyl-CoA synthetase).
This is the only example in the citric acid cycle of substrate-level phosphorylation. Tissues in which gluconeogenesis occurs (the liver and kidney) contain two
isoenzymes of succinate thiokinase, one specific for GDP and the other for ADP. The GTP formed is used for the decarboxylation of oxaloacetate to phosphoenolpyruvate
in gluconeogenesis and provides a regulatory link between citric acid cycle activity and the withdrawal of oxaloacetate for gluconeogenesis. Nongluconeogenic tissues have only the isoenzyme that uses ADP.
When ketone bodies are being metabolized in extrahepatic tissues there is an alternative reaction catalyzed by succinyl-CoA–acetoacetate-CoA transferase (thiophorase)— involving transfer of CoA from succinyl- CoA to acetoacetate, forming acetoacetyl-CoA. The onward metabolism of succinate, leading to the regeneration of oxaloacetate, is the same sequence of chemical reactions as occurs in the β-oxidation of fatty acids: dehydrogenation to form a carbon-carbon double
bond, addition of water to form a hydroxyl group, and a further dehydrogenation to yield the oxo- group of oxaloacetate.
The first dehydrogenation reaction, forming fumarate, is catalyzed by succinate dehydrogenase, which is bound to the inner surface of the inner mitochondrial
membrane. The enzyme contains FAD and iron-sulfur (Fe:S) protein and directly reduces ubiquinone in the respiratory chain. Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate, yielding malate. Malate is converted to oxaloacetate by malate dehydrogenase, a reaction requiring
NAD+. Although the equilibrium of this reaction strongly favors malate, the net flux is toward the direction of oxaloacetate because of the continual removal of
oxaloacetate (either to form citrate, as a substrate for gluconeogenesis, or to undergo transamination to aspartate) and also because of the continual reoxidation of NADH.
TWELVE ATP ARE FORMED PER TURN OF THE CITRIC ACID CYCLE
As a result of oxidations catalyzed by the dehydrogenases of the citric acid cycle, three molecules of NADH and one of FADH2 are produced for each molecule of
acetyl-CoA catabolized in one turn of the cycle. These reducing equivalents are transferred to the respiratory chain, where reoxidation of each NADH results in formation of 3 ATP and reoxidation of FADH2 in formation of 2 ATP. In addition, 1 ATP (or GTP) is formed by substrate-level phosphorylation catalyzed by succinate thiokinase.
VITAMINS PLAY KEY ROLES IN THE CITRIC ACID CYCLE
Four of the B vitamins are essential in the citric acid cycle and therefore in energy-yielding metabolism: (1) riboflavin, in the form of flavin adenine dinucleotide
(FAD), a cofactor in the α-ketoglutarate dehydrogenase complex and in succinate dehydrogenase; (2) niacin, in the form of nicotinamide adenine dinucleotide (NAD),
the coenzyme for three dehydrogenases in the cycle— isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase; (3) thiamin (vitamin
B1), as thiamin diphosphate, the coenzyme for decarboxylation in the α-ketoglutarate dehydrogenase reaction; and (4) pantothenic acid, as part of coenzyme A,
the cofactor attached to “active” carboxylic acid residues such as acetyl-CoA and succinyl-CoA.
THE CITRIC ACID CYCLE PLAYS A PIVOTAL ROLE IN METABOLISM
The citric acid cycle is not only a pathway for oxidation of two-carbon units—it is also a major pathway for interconversion of metabolites arising from transamination
and deamination of amino acids. It also provides the substrates for amino acid synthesis by transamination, as well as for gluconeogenesis and fatty acid synthesis.
Because it functions in both oxidative and synthetic processes, it is amphibolic.
The Citric Acid Cycle Takes Part in Gluconeogenesis, Transamination, & Deamination
All the intermediates of the cycle are potentially glucogenic, since they can give rise to oxaloacetate and thus net production of glucose (in the liver and kidney, the
organs that carry out gluconeogenesis). The key enzyme that catalyzes net transfer out of the cycle into gluconeogenesis is phosphoenolpyruvate carboxykinase, which decarboxylates oxaloacetate to phosphoenolpyruvate, with GTP acting as the donor phosphate. Net transfer into the cycle occurs as a result of several different reactions. Among the most important of such anaplerotic reactions is the formation of oxaloacetate by the carboxylation of pyruvate, catalyzed by pyruvate carboxylase. This reaction is important in maintaining an adequate concentration of oxaloacetate for the condensation reaction with acetyl-CoA. If acetyl- CoA accumulates, it acts both as an allosteric activator of pyruvate carboxylase and as an inhibitor of pyruvate dehydrogenase, thereby ensuring a supply of oxaloacetate.
Lactate, an important substrate for gluconeogenesis, enters the cycle via oxidation to pyruvate and then carboxylation to oxaloacetate.
Aminotransferase (transaminase) reactions form pyruvate from alanine, oxaloacetate from aspartate, and α-ketoglutarate from glutamate. Because these reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of these amino acids. Other amino acids contribute to gluconeogenesis because their carbon skeletons give rise to citric acid cycle intermediates. Alanine, cysteine, glycine, hydroxyproline, serine, threonine, and tryptophan yield pyruvate;
arginine, histidine, glutamine, and proline yield α-ketoglutarate; isoleucine, methionine, and valine yield succinyl-CoA; and tyrosine and phenylalanine yield fumarate.
In ruminants, whose main metabolic fuel is shortchain fatty acids formed by bacterial fermentation, the conversion of propionate, the major glucogenic product of rumen fermentation, to succinyl-CoA via the methylmalonyl-CoA pathway is especially
The Citric Acid Cycle Takes Part in Fatty Acid Synthesis
Acetyl-CoA, formed from pyruvate by the action of pyruvate dehydrogenase, is the major building block for long-chain fatty acid synthesis in nonruminants. (In ruminants, acetyl-CoA is derived directly from acetate.)
Pyruvate dehydrogenase is a mitochondrial enzyme, and fatty acid synthesis is a cytosolic pathway, but the mitochondrial membrane is impermeable to acetyl-
CoA. Acetyl-CoA is made available in the cytosol from citrate synthesized in the mitochondrion, transported into the cytosol and cleaved in a reaction catalyzed by
Regulation of the Citric Acid Cycle Depends Primarily on a Supply of Oxidized Cofactors
In most tissues, where the primary role of the citric acid cycle is in energy-yielding metabolism, respiratory control via the respiratory chain and oxidative phosphorylation regulates citric acid cycle activity. Thus, activity is immediately dependent on the supply of NAD+, which in turn, because of the tight coupling between oxidation and phosphorylation, is dependent on the availability of ADP and hence, ultich16. mately, on the rate of utilization of ATP in chemical and physical work. In addition, individual enzymes of the cycle are regulated. The most likely sites for regulation are the nonequilibrium reactions catalyzed by pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. The
dehydrogenases are activated by Ca2+, which increases in concentration during muscular contraction and secretion, when there is increased energy demand. In a
tissue such as brain, which is largely dependent on carbohydrate to supply acetyl-CoA, control of the citric acid cycle may occur at pyruvate dehydrogenase. Several
enzymes are responsive to the energy status, as shown by the [ATP]/[ADP] and [NADH]/[NAD+] ratios. Thus, there is allosteric inhibition of citrate synthase
by ATP and long-chain fatty acyl-CoA. Allosteric activation of mitochondrial NAD-dependent isocitrate dehydrogenase by ADP is counteracted by ATP and NADH. The α-ketoglutarate dehydrogenase complex is regulated in the same way as is pyruvate dehydrogenase. Succinate dehydrogenase is inhibited by oxaloacetate, and the availability of oxaloacetate, as controlled by malate dehydrogenase, depends on the
[NADH]/[NAD+] ratio. Since the Km for oxaloacetate of citrate synthase is of the same order of magnitude as the intramitochondrial concentration, it is likely that
the concentration of oxaloacetate controls the rate of citrate formation. Which of these mechanisms are important in vivo has still to be resolved.
• The citric acid cycle is the final pathway for the oxidation of carbohydrate, lipid, and protein whose common end-metabolite, acetyl-CoA, reacts with oxaloacetate
to form citrate. By a series of dehydrogenations and decarboxylations, citrate is degraded, releasing reduced coenzymes and 2CO2 and regenerating oxaloacetate.
• The reduced coenzymes are oxidized by the respiratory chain linked to formation of ATP. Thus, the cycle is the major route for the generation of ATP and is located in the matrix of mitochondria adjacent to the enzymes of the respiratory chain and oxidative phosphorylation.
• The citric acid cycle is amphibolic, since in addition to oxidation it is important in the provision of carbon skeletons for gluconeogenesis, fatty acid synthesis, and interconversion of amino acids.
The bulk of ATP used by many cells to maintain homeostasis is produced by the oxidation of pyruvate in the TCA cycle. During this oxidation process, reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2) are generated. The NADH and FADH2 are principally used to drive the processes of oxidative phosphorylation, which are responsible for converting the reducing potential of NADH and FADH2 to the high energy phosphate in ATP.
The fate of pyruvate depends on the cell energy charge. In cells or tissues with a high energy charge pyruvate is directed toward gluconeogenesis, but when the energy charge is low pyruvate is preferentially oxidized to CO2 and H2O in the TCA cycle, with generation of 15 equivalents of ATP per pyruvate. The enzymatic activities of the TCA cycle (and of oxidative phosphorylation) are located in the mitochondrion. When transported into the mitochondrion, pyruvate encounters two principal metabolizing enzymes: pyruvate carboxylase (a gluconeogenic enzyme) and pyruvate dehydrogenase (PDH), the first enzyme of the PDH complex. With a high cell-energy charge coenzyme A (CoA) is highly acylated, principally as acetyl-CoA, and able allosterically to activate pyruvate carboxylase, directing pyruvate toward gluconeogenesis. When the energy charge is low CoA is not acylated, pyruvate carboxylase is inactive, and pyruvate is preferentially metabolized via the PDH complex and the enzymes of the TCA cycle to CO2 and H2O. Reduced NADH and FADH2 generated during the oxidative reactions can then be used to drive ATP synthesis via oxidative phosphorylation.
The PDH complex is comprised of multiple copies of 3 separate enzymes: pyruvate dehydrogenase (20-30 copies), dihydrolipoyl transacetylase (60 copies) and dihydrolipoyl dehydrogenase (6 copies). The complex also requires 5 different coenzymes: CoA, NAD+, FAD+, lipoic acid and thiamine pyrophosphate (TPP). Three of the coenzymes of the complex are tightly bound to enzymes of the complex (TPP, lipoic acid and FAD+) and two are employed as carriers of the products of PDH complex activity (CoA and NAD+). The pathway of PDH oxidation of pyruvate to acetyl-CoA is diagrammed below.
Flow diagram depicting the overall activity of the pyruvate dehydrogenase complex. During the oxidation of pyruvate to CO2 by pyruvate dehydrogenase the electrons flow from pyruvate to the lipoamide moiety of dihydrolipoyl transacetylase then to the FAD cofactor of dihydrolipoyl dehydrogenase and finally to reduction of NAD+ to NADH. The acetyl group is linked to coenzyme A (CoASH) in a high energy thioester bond. The acetyl-CoA then enters the TCA cycle for complete oxidation to CO2 and H2O.
The first enzyme of the complex is PDH itself which oxidatively decarboxylates pyruvate. During the course of the reaction the acetyl group derived from decarboxylation of pyruvate is bound to TPP. The next reaction of the complex is the transfer of the 2--carbon acetyl group from acetyl-TPP to lipoic acid, the covalently bound coenzyme of lipoyl transacetylase. The transfer of the acetyl group from acyl-lipoamide to CoA results in the formation of 2 sulfhydryl (SH) groups in lipoate requiring reoxidation to the disulfide (S-S) form to regenerate lipoate as a competent acyl acceptor. The enzyme dihydrolipoyl dehydrogenase, with FAD+ as a cofactor, catalyzes that oxidation reaction. The final activity of the PDH complex is the transfer of reducing equivalents from the FADH2 of dihydrolipoyl dehydrogenase to NAD+. The fate of the NADH is oxidation via mitochondrial electron transport, to produce 3 equivalents of ATP:
The net result of the reactions of the PDH complex are:
Pyruvate + CoA + NAD+ ------> CO2 + acetyl-CoA + NADH + H+
Regulation of the PDH Complex The reactions of the PDH complex serves to interconnect the metabolic pathways of glycolysis, gluconeogenesis and fatty acid synthesis to the TCA cycle. As a consequence, the activity of the PDH complex is highly regulated by a variety of allosteric effectors and by covalent modification. The importance of the PDH complex to the maintenance of homeostasis is evident from the fact that although diseases associated with deficiencies of the PDH complex have been observed, affected individuals often do not survive to maturity. Since the energy metabolism of highly aerobic tissues such as the brain is dependent on normal conversion of pyruvate to acetyl-CoA, aerobic tissues are most sensitive to deficiencies in components of the PDH complex. Most genetic diseases associated with PDH complex deficiency are due to mutations in PDH. The main pathologic result of such mutations is moderate to severe cerebral lactic acidosis and encephalopathies.
The main regulatory features of the PDH complex are diagrammed below.
Factors regulating the activity of pyruvate dehydrogenase, (PDH). PDH activity is regulated by its' state of phosphorylation, being most active in the dephosphorylated state. Phosphorylation of PDH is catalyzed by a specific PDH kinase. The activity of the kinase is enhanced when cellular energy charge is high which is reflected by an increase in the level of ATP, NADH and acetyl-CoA. Conversely, an increase in pyruvate strongly inhibits PDH kinase. Additional negative effectors of PDH kinase are ADP, NAD+ and CoASH, the levels of which increase when energy levels fall. The regulation of PDH phosphatase is not completely understood but it is known that Mg2+ and Ca2+ activate the enzyme. In adipose tissue insulin increases PDH activity and in cardiac muscle PDH activity is increased by catecholamines.
Two products of the complex, NADH and acetyl-CoA, are negative allosteric effectors on PDH-a, the non-phosphorylated, active form of PDH. These effectors reduce the affinity of the enzyme for pyruvate, thus limiting the flow of carbon through the PDH complex. In addition, NADH and acetyl-CoA are powerful positive effectors on PDH kinase, the enzyme that inactivates PDH by converting it to the phosphorylated PDH-b form. Since NADH and acetyl-CoA accumulate when the cell energy charge is high, it is not surprising that high ATP levels also up-regulate PDH kinase activity, reinforcing down-regulation of PDH activity in energy-rich cells. Note, however, that pyruvate is a potent negative effector on PDH kinase, with the result that when pyruvate levels rise, PDH-a will be favored even with high levels of NADH and acetyl-CoA.
Concentrations of pyruvate which maintain PDH in the active form (PDH-a) are sufficiently high so that, in energy-rich cells, the allosterically down-regulated, high Km form of PDH is nonetheless capable of converting pyruvate to acetyl-CoA. With large amounts of pyruvate in cells having high energy charge and high NADH, pyruvate carbon will be directed to the 2 main storage forms of carbon (glycogen via gluconeogenesis and fat production via fatty acid synthesis) where acetyl-CoA is the principal carbon donor.
Although the regulation of PDH-b phosphatase is not well understood, it
is quite likely regulated to maximize pyruvate oxidation under energy-poor
conditions and to minimize PDH activity under energy-rich conditions.