Basic principles of metabolism: catabolism, anabolism

Basic principles of metabolism: catabolism, anabolism. Common pathways of proteins, carbohydrates and lipids metabolism. Bioenergetics. Krebs cycle, biological oxidation, tissue respiration, oxidative phosphorylation, ATP synthesis.

 

 

BIOMEDICAL IMPORTANCE

The fate of dietary components after digestion and absorption constitutes metabolismthe 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.

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Amphibolic pathways occur at the crossroads of metabolism, acting as links between the anabolic and catabolic pathways, eg, the citric acid cycle.

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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.

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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 bodys main fuel reserve. Acetyl-CoA formed by β-oxidation may undergo several fates:

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(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

OF ORGANIZATION

 

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.

 

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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 processes.

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

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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 aminoacids 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 development.

 

THE CITRIC ACID CYCLE PROVIDES SUBSTRATE FOR THE

RESPIRATORY CHAIN

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.

 

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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.

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The thioester bond of the resultant citryl-CoA is hydrolyzed, releasing citrate and CoASHan 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 complexthiamin diphosphate, lipoate, NAD+, FAD, and CoAand 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-CoAacetoacetate-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 unitsit 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 important.

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

ATP-citrate lyase.

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.

ATP 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 Pyruvate Dehydrogenase (PDH) Complex

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.

 

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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.

 

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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.

 


Bioenergetic processes.

Bioenergetics, or biochemical thermodynamics, is the study of the energy changes accompanying biochemical reactions. Biologic systems are essentially isothermic and use chemical energy to power living processes. How an animal obtains suitable fuel from its food to provide this energy is basic to the understanding of normal nutrition and metabolism. Death from starvation occurs when available energy reserves are depleted, and certain forms of malnutrition are associated with energy imbalance (marasmus). Thyroid hormones control the rate of energy release (metabolic rate), and disease results when they malfunction. Excess storage of surplus energy causes obesity, one of the most common diseases of Western society.

 

FREE ENERGY IS THE USEFUL ENERGY IN A SYSTEM

 

Gibbs change in free energy (ΔG) is that portion of the total energy change in a system that is available for doing workie, the useful energy, also known as the chemical potential.

 

Biologic Systems Conform to the General Laws of Thermodynamics

 

The first law of thermodynamics states that the total energy of a system, including its surroundings, remains constant. It implies that within the total system, energy is neither lost nor gained during any change. However, energy may be transferred from one part of the system to another or may be transformed into another form of energy. In living systems, chemical energy may be transformed into heat or into electrical, radiant, or mechanical energy.

The second law of thermodynamics states that the total entropy of a system must increase if a process is to occur spontaneously. Entropy is the extent of disorder or randomness of the system and becomes maximum as equilibrium is approached. Under conditions of constant temperature and pressure, the relationship between the free energy change (ΔG) of a reacting system and the change in entropy (ΔS) is expressed by the following equation, which combines the two laws of thermodynamics: where ΔH is the change in enthalpy (heat) and T is the absolute temperature.

In biochemical reactions, because ΔH is approximately equal to ΔE, the total change in internal energy of the reaction, the above relationship may be expressed in the following way: If ΔG is negative, the reaction proceeds spontaneously

with loss of free energy; ie, it is exergonic. If, in addition, ΔG is of great magnitude, the reaction goes virtually to completion and is essentially irreversible.

On the other hand, if ΔG is positive, the reaction proceeds only if free energy can be gained; ie, it is endergonic. If, in addition, the magnitude of ΔG is great, the system is stable, with little or no tendency for a reaction to occur. If ΔG is zero, the system is at equilibrium and no net change takes place.

When the reactants are present in concentrations of 1.0 mol/L, ΔG0 is the standard free energy change. For biochemical reactions, a standard state is defined as having a pH of 7.0. The standard free energy change at this standard state is denoted by ΔG0′. The standard free energy change can be calculated from the equilibrium constant Keq. where R is the gas constant and T is the absolute temperature. It is important to note that the actual ΔG may be larger or smaller than ΔG0′ depending on the concentrations of the various reactants, including the solvent, various ions, and proteins.

In a biochemical system, an enzyme only speeds up the attainment of equilibrium; it never alters the final concentrations of the reactants at equilibrium.

 

ENDERGONIC PROCESSES PROCEED BY COUPLING TO EXERGONIC PROCESSES

 

The vital processeseg, synthetic reactions, muscular contraction, nerve impulse conduction, and active transportobtain energy by chemical linkage, or coupling,

to oxidative reactions. In its simplest form, this type of coupling may be represented as shown in Figure 101.

The conversion of metabolite A to metabolite B occurs with release of free energy. It is coupled to another reaction, in which free energy is required to convert metabolite C to metabolite D. The terms exergonic and endergonic rather than the normal chemical terms exothermic and endothermic are used to indicate that a process is accompanied by loss or gain, respectively, of free energy in any form, not necessarily as heat. In practice, an endergonic process cannot exist independently but must be a component of a coupled exergonic- endergonic system where the overall net change is exergonic. The exergonic reactions are termed catabolism (generally, the breakdown or oxidation of fuel molecules), whereas the synthetic reactions that build up substances are termed anabolism. The combined catabolic and anabolic processes constitute metabolism. If the reaction shown in Figure 101 is to go from left to right, then the overall process must be accompanied by loss of free energy as heat. One possible mechanism of coupling could be envisaged if a common obligatory intermediate (I) took part in both reactions, ie, Some exergonic and endergonic reactions in biologic systems are coupled in this way. This type of system has a built-in mechanism for biologic control of the rate of oxidative processes since the common obligatory intermediate allows the rate of utilization of the product of the synthetic path (D) to determine by mass action the rate at which A is oxidized. Indeed, these relationships supply a basis for the concept of respiratory control, the process that prevents an organism from burning out of control. An extension of the coupling concept is provided by dehydrogenation reactions, which are coupled to hydrogenations by an intermediate carrier. An alternative method of coupling an exergonic to an endergonic process is to synthesize a compound of high-energy potential in the exergonic reaction and to incorporate this new compound into the endergonic reaction, thus effecting a transference of free energy from the exergonic to the endergonic pathway.

 

 

 

HIGH-ENERGY PHOSPHATES PLAY A CENTRAL ROLE IN ENERGY CAPTURE AND TRANSFER

 

In order to maintain living processes, all organisms must obtain supplies of free energy from their environment. Autotrophic organisms utilize simple exergonic

processes; eg, the energy of sunlight (green plants), the reaction Fe2+ → Fe3+ (some bacteria). On the other hand, heterotrophic organisms obtain free energy by coupling their metabolism to the breakdown of complex organic molecules in their environment. In all these organisms, ATP plays a central role in the transference of free energy from the exergonic to the endergonic processes . ATP is a nucleoside triphosphate containing adenine, ribose, and three phosphate groups. In its reactions in the cell, it functions as the Mg2+ complex .

The importance of phosphates in intermediary metabolism became evident with the discovery of the role of ATP, adenosine diphosphate (ADP), and inorganic phosphate (Pi) in glycolysis .

 

High-Energy Phosphates Are Designated by ~ P

 

The symbol ~ P indicates that the group attached to the bond, on transfer to an appropriate acceptor, results in transfer of the larger quantity of free energy. For this reason, the term group transfer potential is preferred by some to high-energy bond. Thus, ATP contains two high-energy phosphate groups and ADP contains one, whereas the phosphate in AMP (adenosine monophosphate) is of the low-energy type, since it is a normal ester link.

 

 

HIGH-ENERGY PHOSPHATES ACT AS THE ENERGY CURRENCY OF THE CELL

 

ATP is able to act as a donor of high-energy phosphate to form those compounds. Likewise, with the necessary enzymes, ADP can accept high-energy phosphate to form ATP from those compounds above ATP in the table. In effect, an ATP/ ADP cycle connects those processes that generate ~ P to those processes that utilize ~ P, continuously consuming and regenerating ATP.

 

 

This occurs at a very rapid rate, since the total ATP/ADP pool is extremely small and sufficient to maintain an active tissue for only a few seconds.

There are three major sources of ~ P taking part in energy conservation or energy capture:

(1) Oxidative phosphorylation: The greatest quantitative source of ~ P in aerobic organisms. Free energy O2 within mitochondria.

(2) Glycolysis: A net formation of two ~ P results from the formation of lactate from one molecule of glucose, generated in two reactions catalyzed by phosphoglycerate kinase and pyruvate kinase, respectively.

(3) The citric acid cycle: One ~ P is generated directly in the cycle at the succinyl thiokinase step.

 

 

 

Biologic Oxidation

 

Chemically, oxidation is defined as the removal of electrons and reduction as the gain of electrons. Thus, oxidation is always accompanied by reduction of an electron acceptor. This principle of oxidation-reduction applies equally to biochemical systems and is an important concept underlying understanding of the nature of biologic oxidation. Note that many biologic oxidations can take place without the participation of molecular oxygen, eg, dehydrogenations. The life of higher animals is absolutely dependent upon a supply of oxygen for respiration, the process by which cells derive energy in the form of ATP from the controlled reaction of hydrogen with oxygen to form water. In addition, molecular oxygen is incorporated into a variety of substrates by enzymes designated as oxygenases; many drugs, pollutants, and chemical carcinogens (xenobiotics) are metabolized

by enzymes of this class, known as the cytochrome P450 system. Administration of oxygen can be lifesaving in the treatment of patients with respiratory

or circulatory failure.

 

FREE ENERGY CHANGES CAN BE EXPRESSED IN TERMS OF REDOX POTENTIA

 

In reactions involving oxidation and reduction, the free energy change is proportionate to the tendency of reactants to donate or accept electrons. Thus, in addition to expressing free energy change in terms of ΔG0′, it is possible, in an analogous manner, to express it numerically as an oxidation-reduction or redox potential (E′0). The redox potential of a system (E0) is usually compared with the potential of the hydrogen electrode (0.0 volts at pH 0.0). However, for biologic

systems, the redox potential (E′0)is normally expressedat pH 7.0, at which pH the electrode potential of the hydrogen electrode is −0.42 volts. The redox potentials of some redox systems of special interest in mammalian biochemistry are shown in Table 111. The relative positions of redox systems in the table allows prediction of the direction of flow of electrons from one redox couple

to another. Enzymes involved in oxidation and reduction are called oxidoreductases and are classified into four groups: oxidases, dehydrogenases, hydroperoxidases, and oxygenases.

 

OXIDASES USE OXYGEN AS A HYDROGEN ACCEPTOR

Oxidases catalyze the removal of hydrogen from a substrate using oxygen as a hydrogen acceptor.* They form water or hydrogen peroxide as a reaction product.

Some Oxidases Contain Copper

Cytochrome oxidase is a hemoprotein widely distributed in many tissues, having the typical heme prosthetic group present in myoglobin, hemoglobin, and other cytochromes. It is the terminal component of the chain of respiratory carriers found in mitochondria and transfers electrons resulting from the oxidation of substrate molecules by dehydrogenases to their final acceptor, oxygen. The enzyme is poisoned by carbon monoxide, cyanide, and hydrogen sulfide. It has also been termed cytochrome a3. It is now known that cytochromes a and a3 are combined in a single protein, and the complex is known as cytochrome aa3. It contains two molecules of heme, each having one Fe atom that oscillates between Fe3+ and Fe2+ during oxidation and reduction. Furthermore, two atoms of Cu are present, each associated with a heme unit.

 

 

Other Oxidases Are Flavoproteins

Flavoprotein enzymes contain flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as prosthetic groups. FMN and FAD are formed in the body from the vitamin riboflavin. FMN and FAD are usually tightlybut not covalentlybound to their respective apoenzyme proteins. Metalloflavoproteins contain one or more metals as essential cofactors. Examples of flavoprotein enzymes include L-amino acid oxidase, an FMN-linked enzyme found in kidney with general specificity for the oxidative deamination of the naturally occurring L-amino acids; xanthine oxidase, which contains molybdenum and plays an important role in the conversion of purine bases to uric acid, and is of particular significance in uricotelic animals; and aldehyde dehydrogenase, an FAD-linked enzyme present in mammalian livers, which contains molybdenum and nonheme iron and acts upon aldehydes and N-heterocyclic substrates. The mechanisms of oxidation and reduction of these enzymes are complex. Evidence suggests a two-step reaction as shown.

DEHYDROGENASES CANNOT USE OXYGEN AS A HYDROGEN ACCEPTOR

 

There are a large number of enzymes in this class. They perform two main functions:

(1) Transfer of hydrogen from one substrate to another in a coupled oxidation-reduction reaction. These dehydrogenases are specific for their substrates but often utilize common coenzymes or hydrogen carriers, eg, NAD+. Since the reactions are reversible, these properties enable reducing equivalents to be freely transferred within the cell. This type of reaction, which enables one substrate to be oxidized at the expense of another, is particularly useful in enabling oxidative

 

 

 

processes to occur in the absence of oxygen, such as during the anaerobic phase of glycolysis.

(2) As components in the respiratory chain of electron transport from substrate to oxygen.

Many Dehydrogenases Depend on Nicotinamide Coenzymes

These dehydrogenases use nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+)or bothand are formed in the body from the vitamin niacin. The coenzymes are reduced by the specific substrate of the dehydrogenase and reoxidized by a suitable electron acceptor .They may freely and reversibly dissociate from their respective apoenzymes. Generally, NAD-linked dehydrogenases catalyze oxidoreduction reactions in the oxidative pathways of metabolism, particularly in glycolysis, in the citric acid cycle, and in the respiratory chain of mitochondria. NADP-linked dehydrogenases are found characteristically in reductive syntheses, as in the extramitochondrial pathway of fatty acid synthesis and steroid synthesisand also in the pentose phosphate pathway.

 

 

Other Dehydrogenases Depend on Riboflavin

 

The flavin groups associated with these dehydrogenases are similar to FMN and FAD occurring in oxidases. They are generally more tightly bound to their apoenzymes than are the nicotinamide coenzymes. Most of the riboflavin-linked dehydrogenases are concerned with electron transport in (or to) the respiratory chain. NADH dehydrogenase acts as a carrier of electrons between NADH and the components of higher redox potential. Other dehydrogenases such as succinate dehydrogenase, acyl-CoA dehydrogenase, and mitochondrial glycerol-3-phosphate dehydrogenase transfer reducing equivalents directly from the substrate to the respiratory chain. Another role of the flavin-dependent dehydrogenases is in the dehydrogenation (by dihydrolipoyl dehydrogenase) of reduced lipoate, an intermediate in the oxidative decarboxylation of pyruvate and α-ketoglutarate. The electron-transferring flavoprotein is an intermediary

carrier of electrons between acyl-CoA dehydrogenase and the respiratory chain

 

Oxidative Phosphorylation

Aerobic organisms are able to capture a far greater proportion of the available free energy of respiratory substrates than anaerobic organisms. Most of this takes place inside mitochondria, which have been termed the powerhouses of the cell. Respiration is coupled to the generation of the high-energy intermediate, ATP, by oxidative phosphorylation, and the chemiosmotic theory offers insight into how this is accomplished. A number of drugs (eg, amobarbital) and poisons (eg,cyanide, carbon monoxide) inhibit oxidative phosphorylation, usually with fatal consequences. Several inherited defects of mitochondria involving components of the respiratory chain and oxidative phosphorylation have been reported. Patients present with myopathy and encephalopathy and often have lactic acidosis.

 

 

SPECIFIC ENZYMES ACT AS MARKERS OF COMPARTMENTS

SEPARATED BY THE MITOCHONDRIAL MEMBRANES

 

Mitochondria have an outer membrane that is permeable to most metabolites, an inner membrane that is selectively permeable, and a matrix within. The outer membrane is characterized by the presence of various enzymes, including acyl-CoA synthetase and glycerolphosphate acyltransferase. Adenylyl kinase and creatine kinase are found in the intermembrane space. The phospholipid cardiolipin is concentrated in the inner membrane together with the enzymes

of the respiratory chain.

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THE RESPIRATORY CHAIN COLLECTS & OXIDIZES REDUCING EQUIVALENTS

 

Most of the energy liberated during the oxidation of carbohydrate, fatty acids, and amino acids is made available within mitochondria as reducing equivalents (H or electrons). Mitochondria contain the respiratory chain, which collects and transports reducing equivalents directing them to their final reaction with oxygen to form water, the machinery for trapping the liberated free energy as high-energy phosphate, and the enzymes of β-oxidation and of the citric acid cycle) that produce most of the reducing equivalents.

Components of the Respiratory Chain Are Arranged in Order of Increasing

Redox Potential

 

Hydrogen and electrons flow through the respiratory chain through a redox span of 1.1 V from NAD+/NADH to O2/2H2O.

 

The respiratory chain consists of a number of redox carriers that proceed from the NAD-linked dehydrogenase systems, through flavoproteins and cytochromes, to molecular oxygen. Not all substrates are linked to the respiratory chain through NAD-specific dehydrogenases; some, because their redox potentials are more positive (eg, fumarate/succinate), are linked directly to flavoprotein dehydrogenases, which in turn are linked to the cytochromes of the respiratory chain.

Ubiquinone or Q (coenzyme Q) (Figure 125) links the flavoproteins to cytochrome b, the member of the cytochrome chain of lowest redox potential.

Q exists in the oxidized quinone or reduced quinol form under aerobic or anaerobic conditions, respectively. The structure of Q is very similar to that of vitamin K and vitamin E and of plastoquinone, found in chloroplasts. Q acts as a mobile component of the respiratory chain that collects reducing equivalents from the more fixed flavoprotein complexes and passes them on to the cytochromes. An additional component is the iron-sulfur protein (FeS; nonheme iron) . It is associated with the flavoproteins (metalloflavoproteins) and with cytochrome b. The sulfur and iron are thought to take part in the oxidoreduction mechanism between flavin and Q, which involves only a single e− change, the iron atom undergoing oxidoreduction between Fe2+ and

Fe3+.

Pyruvate and α-ketoglutarate dehydrogenase have complex systems involving lipoate and FAD prior to the passage of electrons to NAD, while electron transch12.fers from other dehydrogenases, eg, L(+)-3-hydroxyacyl-CoA dehydrogenase, couple directly with NAD. The reduced NADH of the respiratory chain is in turn oxidized by a metalloflavoprotein enzymeNADH dehydrogenase. This enzyme contains FeS and FMN, is tightly bound to the respiratory chain, and passes reducing equivalents on to Q.

 

THE RESPIRATORY CHAIN PROVIDES MOST OF THE ENERGY CAPTURED DURING CATABOLISM

ADP captures, in the form of high-energy phosphate, a significant proportion of the free energy released by catabolic processes. The resulting ATP has been called the energy currency of the cell because it passes on this free energy to drive those processes requiring energy. There is a net direct capture of two high-energy phosphate groups in the glycolytic reactions, equivalent to approximately 103.2 kJ/mol of glucose. (In vivo, ΔG for the synthesis of ATP from ADP has been calculated as approximately 51.6 kJ/mol. (It is greater than ΔG0′ for the hydrolysis of ATP as given in Table 101, which is obtained under standard

 

 

 

Respiratory Control Ensures a Constant Supply of ATP

 

The rate of respiration of mitochondria can be controlled by the availability of ADP. This is because oxidation and phosphorylation are tightly coupled; ie, oxidation cannot proceed via the respiratory chain without concomitant phosphorylation of ADP shows the five conditions controlling the rate of respiration in mitochondria. Most cells in the resting state are in state 4, and respiration is controlled by the availability of ADP. When work is performed, ATP is converted to ADP, allowing more respiration to occur, which in turn replenishes the store of ATP. Under certain conditions, the concentration of inorganic phosphate can also affect the rate of functioning of the respiratory chain. As respiration increases (as in exercise), the cell approaches state 3 or state 5 when either the capacity of the respiratory chain becomes saturated or the PO2 decreases below the Km for cytochrome a3. There is also the possibility that the ADP/ATP transporter , which facilitates entry of cytosolic ADP into and ATP out of the mitochondrion, becomes ratelimiting. Thus, the manner in which biologic oxidative processes allow the free energy resulting from the oxidation of foodstuffs to become available and to be captured is stepwise, efficient (approximately 68%), and controlledrather than explosive, inefficient, and uncontrolled, as in many nonbiologic processes. The remaining free energy that is not captured as high-energy phosphate is liberated as heat. This need not be considered wasted, since it ensures that the respiratory system as a whole is sufficiently exergonic to be removed from equilibrium, allowing continuous unidirectional flow and constant provision of ATP. It also contributes to maintenance of body temperature.

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MANY POISONS INHIBIT THE RESPIRATORY CHAIN

Formation and transfer of disulphide bonds in living cells

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Much information about the respiratory chain has been obtained by the use of inhibitors, and, conversely, this has provided knowledge about the mechanism of action of several poisons . They may be classified as inhibitors of the respiratory chain, inhibitors of oxidative phosphorylation, and uncouplers of oxidative phosphorylation. Barbiturates such as amobarbital inhibit NAD-linked dehydrogenases by blocking the transfer from FeS to Q. At sufficient dosage, they are fatal in vivo.

 

Antimycin A and dimercaprol inhibit the respiratory chain between cytochrome b and cytochrome c. The classic poisons H2S, carbon monoxide, and cyanide inhibit cytochrome oxidase and can therefore totally arrest respiration. Malonate is a competitive inhibitor of succinate dehydrogenase. Atractyloside inhibits oxidative phosphorylation by inhibiting the transporter of ADP into and ATP out of the mitochondrion .

The action of uncouplers is to dissociate oxidation in the respiratory chain from phosphorylation. These compounds are toxic in vivo, causing respiration to become uncontrolled, since the rate is no longer limited by the concentration of ADP or Pi. The uncoupler that has been used most frequently is 2,4-dinitrophenol, but other compounds act in a similar manner. The antibiotic oligomycin completely blocks oxidation and phosphorylation by acting on a step in phosphorylation.

 

THE CHEMIOSMOTIC THEORY EXPLAINS THE MECHANISM OF OXIDATIVE PHOSPHORYLATION

 

Mitchells chemiosmotic theory postulates that the energy from oxidation of components in the respiratory chain is coupled to the translocation of hydrogen ions (protons, H+) from the inside to the outside of the inner mitochondrial membrane. The electrochemical potential difference resulting from the asymmetric distribution of the hydrogen ions is used to drive the mechanism responsible for the formation of ATP .

 

The Respiratory Chain Is a Proton Pump

Each of the respiratory chain complexes I, III, and IV acts as a proton pump. The inner membrane is impermeable to ions in general but particularly to protons, which accumulate outside the membrane, creating an electrochemical potential difference across the membrane (ΔμH +).This consists of a chemical potential (difference in pH) and an electrical potential.

A Membrane-Located ATP Synthase Functions as a Rotary Motor to Form ATP

The electrochemical potential difference is used to drive a membrane-located ATP synthase which in the presence of Pi + ADP forms ATP . Scattered over the surface of the inner membrane are the phosphorylating complexes, ATP synthase, responsible for the production of ATP . These consist of several protein subunits, collectively known as F1, which project into the matrix and which contain the phosphorylation mechanism . These subsub unitsare attached to a membrane protein complex known as F0, which also consists of several protein subunits.

F0 spans the membrane and forms the proton channel. The flow of protons through F0 causes it to rotate, driving the production of ATP in the F1 complex. Estimates suggest that for each NADH oxidized, complex I translocates four protons and complexes III and IV translocate 6 between them. As four protons are taken into the mitochondrion for each ATP exported, the P:O ratio would not necessarily be a complete integer, ie, 3, but possibly 2.5. However, for simplicity,

a value of 3 for the oxidation of NADH + H+ and 2 for the oxidation of FADH2 will continue to be used throughout this text.

 

Experimental Findings Support the Chemiosmotic Theory

 

(1) Addition of protons (acid) to the external medium of intact mitochondria leads to the generation of ATP.

(2) Oxidative phosphorylation does not occur in soluble systems where there is no possibility of a vectorial ATP synthase. A closed membrane must be present in

order to achieve oxidative phosphorylation .

(3) The respiratory chain contains components organized in a sided manner (transverse asymmetry) as required by the chemiosmotic theory.

 

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