Basic principles of metabolism: catabolism, anabolism. Common pathways of proteins,
carbohydrates and lipids transformation. Investigation of Krebs cycle functioning.
Metabolism is the set of life-sustaining chemical transformations within the cells of living organisms. These enzyme-catalyzed reactions allow organisms to grow and reproduce, maintain their structures, and respond to their environments. The word metabolism can also refer to all chemical reactions that occur in living organisms, including digestion and the transport of substances into and between different cells, in which case the set of reactions within the cells is called intermediary metabolism or intermediate metabolism.
The term metabolism is derived from the Greek –
"Metabolismos" for "change", or "overthrow". The history of the scientific study of
metabolism spans several centuries and has moved from examining whole animals
in early studies, to examining individual metabolic reactions in modern
biochemistry. The first controlled experiments in human metabolism were published by Santorio Santorioin
In these early studies, the mechanisms of these metabolic processes had not been identified and a vital force was thought to animate living tissue. In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that fermentation was catalyzed by substances within the yeast cells he called "ferments". He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells."] This discovery, along with the publication by Friedrich Wöhler in 1828 of the chemical synthesis of urea, notable for being the first organic compound prepared from wholly inorganic precursors, proved that the organic compounds and chemical reactions found in cells were no different in principle than any other part of chemistry.
It was the discovery of enzymes at the beginning of the 20th century by Eduard Buchner that separated the study of the chemical reactions of metabolism from the biological study of cells, and marked the beginnings of biochemistry. The mass of biochemical knowledge grew rapidly throughout the early 20th century. One of the most prolific of these modern biochemists wasHans Krebs who made huge contributions to the study of metabolism. He discovered the urea cycle and later, working with Hans Kornberg, the citric acid cycle and the glyoxylate cycle. Modern biochemical research has been greatly aided by the development of new techniques such as chromatography, X-ray diffraction, NMR spectroscopy, radioisotopic labelling, electron microscopy andmolecular dynamics simulations. These techniques have allowed the discovery and detailed analysis of the many molecules and metabolic pathways in cells.
Metabolism is a term that is used to describe all chemical reactions involved in maintaining the living state of the cells and the organism. Metabolism can be conveniently divided into two categories:
· Catabolism - the breakdown of molecules to obtain energy
· Anabolism - the synthesis of all compounds needed by the cells
Anabolism is the set of constructive metabolic processes where the energy released by catabolism is used to synthesize complex molecules. In general, the complex molecules that make up cellular structures are constructed step-by-step from small and simple precursors. Anabolism involves three basic stages. Firstly, the production of precursors such as amino acids, monosaccharides,isoprenoids and nucleotides, secondly, their activation into reactive forms using energy from ATP, and thirdly, the assembly of these precursors into complex molecules such as proteins, polysaccharides, lipids and nucleic acids.
Metabolism refers to the highly integrated network of chemical reactions by which living cells grow and sustain themselves. This network is composed of two major types of pathways: anabolism and catabolism. Anabolism uses energy stored in the form of adenosine triphosphate (ATP) to build larger molecules from smaller molecules. Catabolic reactions degrade larger molecules in order to produce ATP and raw materials for anabolic reactions.
Together, these two general metabolic networks have three major functions:
(1) to extract energy from nutrients or solar energy;
(2) to synthesize the building blocks that make up the large molecules of life: proteins, fats, carbohydrates, nucleic acids, and combinations of these substances;
(3) to synthesize and degrade molecules required for special functions in the cell.
These reactions are controlled by enzymes, protein catalysts that increase the speed of chemical reactions in the cell without themselves being changed. Each enzyme catalyzes a specific chemical reaction by acting on a specific substrate, or raw material. Each reaction is just one in a sequence of catalyticsteps known as metabolic pathways. These sequences may be composed of up to20 enzymes, each one creating a product that becomes the substrate--or raw material--for the subsequent enzyme. Often, an additional molecule called a coenzyme is required for the enzyme to function. For example, some coenzymes accept an electron that is released from the substrate during the enzymatic reaction. Most of the water-soluble vitamins of the B complex serve as coenzymes;riboflavin (Vitamin B2) for example, is a precursor of the coenzyme flavine adenine dinucleotide, while pantothenate is a component of coenzyme A, an important intermediate metabolite.
The series of products created by the sequential enzymatic steps of anabolismor catabolism are called metabolic intermediates, or metabolites. Each steprepresents a small change in the molecule, usually the removal, transfer, oraddition of a specific atom, molecule, or group of atoms that serves as a functional group, such as the amino groups (-NH2) of proteins.
Most such metabolic pathways are linear, that is, they begin with a specificsubstrate and end with a specific product. However, some pathways, such as the Krebs cycle, are cyclic. Often, metabolic pathways also have branches thatfeed into or out of them. The specific sequences of intermediates in the pathways of cell metabolism are called intermediary metabolism.
Among the many hundreds of chemical reactions there are only a few that are central to the activity of the cell, and these pathways are identical in mostforms of life.
All reactions of metabolism, however, are part of the overall goal of the organism to maintain its internal orderliness, whether that organism is a singlecelled protozoan or a human. Organisms maintain this orderliness by removingenergy from nutrients or sunlight and returning to their environment an equal amount of energy in a less useful form, mostly heat. This heat becomes dissipated throughout the rest of the organism's environment.
According to the first law of thermodynamics, in any physical or chemical change, the total amount of energy in the universe remains constant, that is, energy cannot be created or destroyed. Thus, when the energy stored in nutrientmolecules is released and captured in the form of ATP, some energy is lost as heat. But the total amount of energy is unchanged.
The second law of thermodynamics states that physical and chemical changes proceed in such a direction that useful energy undergoes irreversible degradation into a randomized form--entropy. The dissipation of energy during metabolism represents an increase in the randomness, or disorder, of the organism's environment. Because this disorder is irreversible, it provides the driving force and direction to all metabolic enzymatic reactions.
Even in the simplest cells, such as bacteria, there are at least a thousand such reactions. Regardless of the number, all cellular reactions can be classified as one of two types of metabolism: anabolism and catabolism. These reactions, while opposite in nature, are linked through the common bond of energy.Anabolism, or biosynthesis, is the synthetic phase of metabolism during which small building block molecules, or precursors, are built into large molecular components of cells, such as carbohydrates and proteins.
Catabolic reactions are used to capture and save energy from nutrients, as well as to degrade larger molecules into smaller, molecular raw materials for reuse by the cell. The energy is stored in the form of energy-rich ATP, whichpowers the reactions of anabolism. The useful energy of ATP is stored in theform of a high-energy bond between the second and third phosphate groups of ATP. The cell makes ATP by adding a phosphate group to the molecule adenosinediphosphate (ADP). Therefore, ATP is the major chemical link between the energy-yielding reactions of catabolism, and the energy-requiring reactions of anabolism.
In some cases, energy is also conserved as energy-rich hydrogen atoms in thecoenzyme nicotinamide adenine dinucleotide phosphate in the reduced form of NADPH. The NADPH can then be used as a source of high-energy hydrogen atoms during certain biosynthetic reactions of anabolism.
In addition to the obvious difference in the direction of their metabolic goals, anabolism and catabolism differ in other significant ways. For example, the various degradative pathways of catabolism are convergent. That is, many hundreds of different proteins, polysaccharides, and lipids are broken down into relatively few catabolic end products. The hundreds of anabolic pathways,however, are divergent. That is, the cell uses relatively few biosynthetic precursor molecules to synthesize a vast number of different proteins, polysaccharides, and lipids.
The opposing pathways of anabolism and catabolism may also use different reaction intermediates or different enzymatic reactions in some of the steps. Forexample, there are 11 enzymatic steps in the breakdown of glucose into pyruvic acid in the liver. But the liver uses only nine of those same steps in thesynthesis of glucose, replacing the other two steps with a different set ofenzyme-catalyzed reactions. This occurs because the pathway to degradation ofglucose releases energy, while the anabolic process of glucose synthesis requires energy. The two different reactions of anabolism are required to overcome the energy barrier that would otherwise prevent the synthesis of glucose.
Another reason for having slightly different pathways is that the corresponding anabolic and catabolic routes must be independently regulated. Otherwise,if the two phases of metabolism shared the exact pathway (only in reverse) aslowdown in the anabolic pathway would slow catabolism, and vice versa.
Some reactions can be either catabolic or anabolic, depending on the circumstances. Such reactions are called amphibolic reactions. Many of the reactions interconverting the “simple molecules” fall in this category.
Catabolic and anabolic pathways are interrelated in three ways:
Matter (catabolic pathways furnish the precursor compounds for anabolism. Energy (catabolic pathways furnish the energy to “drive” anabolism). Electrons (catabolic pathways furnish the reducing power for anabolism).
Linear pathways convert one compound through a series of intermediates to another compound. An example would be glycolysis, where glucose is converted to pyruvate.
Branched pathways may either be divergent (an intermediate can enter several linear pathways to different end products) or convergent (several precursors can give rise to a common intermediate). Biosynthesis of purines and of some amino acids are examples of divergent pathways. There is usually some regulation at the branch point. The conversion of various carbohydrates into the glycolytic pathway would be an example of convergent pathways.
In a cyclic pathway, intermediates are regenerated, and so some intermediates act in a catalytic fashion. In this illustration, the cyclic pathway carries out the net conversion of X to Z. The Tricarboxylic Acid Cycle is an example of a cyclic pathway.
A pool of compounds in equilibrium with each other provides the intermediates for converting compounds to a variety of products, depending on what is fed “into” the pool and what is “withdrawn” from the pool. The phosphogluconate pathway is an example of such a pool of intermediates. The pathway can convert glucose to CO2, hexoses to pentoses, pentoses to hexoses, pentoses to trioses, etc. depending on what the cell requires in a particular situation. NADPH as a source of reducing power for anabolic reactions is also a main product of the phosphogluconate pathway.
Organisms differ in how many of the molecules in their cells they can construct for themselves. Autotrophs such as plants can construct the complex organic molecules in cells such as polysaccharides and proteins from simple molecules like carbon dioxide and water. Heterotrophs, on the other hand, require a source of more complex substances, such as monosaccharides and amino acids, to produce these complex molecules. Organisms can be further classified by ultimate source of their energy: photoautotrophs and photoheterotrophs obtain energy from light, whereas chemoautotrophs and chemoheterotrophs obtain energy from inorganic oxidation reactions.
Metabolism is closely linked to nutrition and the availability of nutrients. Bioenergetics is a term which describes the biochemical or metabolic pathways by which the cell ultimately obtains energy. Energy formation is one of the vital components of metabolism.
The speed of metabolism, the metabolic rate, influences how much food an organism will require, and also affects how it is able to obtain that food.
A striking feature of metabolism is the similarity of the basic metabolic pathways and components between even vastly different species. For example, the set of carboxylic acids that are best known as the intermediates in the citric acid cycle are present in all known organisms, being found in species as diverse as the unicellular bacterium Escherichia coli and huge multicellular organisms.
Nutrition is the key to metabolism. The pathways of metabolism rely upon nutrients that they breakdown in order to produce energy. This energy in turn is required by the body to synthesize new proteins, nucleic acids (DNA, RNA) etc.
Nutrients in relation to metabolism encompass bodily requirement for various substances, individual functions in body, amount needed, level below which poor health results etc.
Essential nutrients supply energy (calories) and supply the necessary chemicals which the body itself cannot synthesize. Food provides a variety of substances that are essential for the building, upkeep, and repair of body tissues, and for the efficient functioning of the body.
The diet needs essential nutrients like carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, and around 20 other inorganic elements. The major elements are supplied incarbohydrates, lipids, and protein. In addition, vitamins, minerals and water are necessary.
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.
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.
Foods supply carbohydrates in three forms: starch, sugar, and cellulose (fiber). Starches and sugars form major and essential sources of energy for humans. Fibers contribute to bulk in diet.
Body tissues depend on glucose for all activities. Carbohydrates and sugars yield glucose by digestion or metabolism.Most people consume around half of their diet as carbohydrates.
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.
Fats are concentrated sources of energy. They produce twice as much energy as either carbohydrates or protein on a weight basis.
Carbohydrate catabolism is the breakdown of carbohydrates into smaller units. Carbohydrates are usually taken into cells once they have been digested intomonosaccharides. Once inside, the major route of breakdown is glycolysis, where sugars such as glucose and fructose are converted into pyruvate and some ATP is generated.
Pyruvate is an intermediate in several metabolic pathways, but the majority is converted to acetyl-CoA and fed into the citric acid cycle. Although some more ATP is generated in the citric acid cycle, the most important product is NADH, which is made from NAD+ as the acetyl-CoA is oxidized. This oxidation releases carbon dioxide as a waste product. In anaerobic conditions, glycolysis produces lactate, through the enzyme lactate dehydrogenase re-oxidizing NADH to NAD+ for re-use in glycolysis. An alternative route for glucose breakdown is the pentose phosphate pathway, which reduces the coenzyme NADPH and produces pentose sugars such asribose, the sugar component of nucleic acids.
Fats are catabolised by hydrolysis to free fatty acids and glycerol. The glycerol enters glycolysis and the fatty acids are broken down bybeta oxidation to release acetyl-CoA, which then is fed into the citric acid cycle. Fatty acids release more energy upon oxidation than carbohydrates because carbohydrates contain more oxygen in their structures.
Amino acids are either used to synthesize proteins and other biomolecules, or oxidized to urea and carbon dioxide as a source of energy. The oxidation pathway starts with the removal of the amino group by a transaminase. The amino group is fed into the urea cycle, leaving a deaminated carbon skeleton in the form of a keto acid. Several of these keto acids are intermediates in the citric acid cycle, for example the deamination of glutamate forms α-ketoglutarate. The glucogenic amino acids can also be converted into glucose, through gluconeogenesis .
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.
Proteins are the main tissue builders in the body. They are part of every cell in the body. Proteins help in cell structure, functions, haemoglobin formation to carry oxygen, enzymes to carry out vital reactions and a myriad of other functions in the body. Proteins are also vital in supplying nitrogen for DNA and RNA genetic material and energy production.
Catabolism can be broken down into 3 main stages.
Stage 1 – Stage of Digestion
The large organic molecules like proteins, lipids and polysaccharides are digested into their smaller components outside cells. This stage acts on starch, cellulose or proteins that cannot be directly absorbed by the cells and need to be broken into their smaller units before they can be used in cell metabolism.
Digestive enzymes include glycoside hydrolases that digest polysaccharides into monosaccharides or simple sugars.
The primary enzyme involved in protein digestion is pepsin which catalyzes the nonspecific hydrolysis of peptide bonds at an optimal pH of 2. In the lumen of the small intestine, the pancreas secretes zymogens of trypsin, chymotrypsin, elastase etc. These proteolytic enzymes break the proteins down into free amino acids as well as dipeptides and tripeptides. The free amino acids as well as the di and tripeptides are absorbed by the intestinal mucosa cells which subsequently are released into the blood stream where they are absorbed by other tissues.
The amino acids and sugars are then pumped into cells by specific active transport proteins.
Stage 2 – Release of energy
Once broken down these molecules are taken up by cells and converted to yet smaller molecules, usually acetyl coenzyme A (acetyl-CoA), which releases some energy.
Stage 3 - The acetyl group on the CoA is oxidised to water and carbon dioxide in the citric acid cycle and electron transport chain, releasing the energy that is stored by reducing the coenzyme nicotinamide adenine dinucleotide (NAD+) into NADH.
When complex carbohydrates are broken they form simple sugars or monosaccharides. This is taken up by the cells. Once inside these sugars undergo glycolysis, where sugars such as glucose and fructose are converted into pyruvate and some ATP is generated. Pyruvate is an intermediate in several metabolic pathways, but the majority is converted to acetyl-CoA and fed into the citric acid cycle or the Kreb’s cycle.
Within the citric acid cycle more ATP is generated by the monosaccharides. The most important product is NADH, which is made from NAD+ as the acetyl-CoA is oxidized. This oxidation releases carbon dioxide as a waste product.
When there is no oxygen, glycolysis produces lactate, through the enzyme lactate dehydrogenase, re-oxidizing NADH to NAD+ for re-use in glycolysis.
Glucose can also be broken down by pentose phosphate pathway, which reduces the coenzyme NADPH and produces pentose sugars such as ribose, the sugar component of nucleic acids.
Proteins are broken down into amino acids. Amino acids are either used to synthesize proteins and other biomolecules, or oxidized to urea and carbon dioxide as a source of energy.
In the process of oxidation, first the amino group is removed by a transaminase. The amino group is fed into the urea cycle, leaving a deaminated carbon skeleton in the form of a keto acid.
These keto acids enter the citric acid cycle. Glutamate, for example, forms α-ketoglutarate. Some of the amines may also be converted into glucose, through gluconeogenesis.
Some proteins are incredibly stable, others are very short lived. The short lived proteins usually play important metabolic roles. The short life times of these proteins allow the cell to rapidly adjust to changes in the metabolic state of the cell.
Fats are catabolised by hydrolysis to free fatty acids and glycerol. The glycerol enters glycolysis and the fatty acids are broken down by beta oxidation to release acetyl-CoA. This acetyl co-A reaches the citric acid cycle next. Fatty acids release more energy upon oxidation than carbohydrates because carbohydrates contain more oxygen in their structures.
The chemical reactions of metabolism are organized into metabolic pathways. These allow the basic chemicals from nutrition to be transformed through a series of steps into another chemical, by a sequence of enzymes.
Enzymes are crucial to metabolism because they allow organisms to drive desirable reactions that require energy. These reactions also are coupled with those that release energy. As enzymes act as catalysts they allow these reactions to proceed quickly and efficiently. Enzymes also allow the regulation of metabolic pathways in response to changes in the cell's environment or signals from other cells.
Each metabolic pathway consists of a series of biochemical reactions that are connected by their intermediates: the products of one reaction are the substrates for subsequent reactions, and so on. Metabolic pathways are often considered to flow in one direction. Although all chemical reactions are technically reversible, conditions in the cell are often such that it is thermodynamically more favorable for flux to flow in one direction of a reaction. For example, one pathway may be responsible for the synthesis of a particular amino acid, but the breakdown of that amino acid may occur via a separate and distinct pathway. One example of an exception to this "rule" is the metabolism of glucose. Glycolysis results in the breakdown of glucose, but several reactions in the glycolysis pathway are reversible and participate in the re-synthesis of glucose (gluconeogenesis).
· Glycolysis was the first metabolic pathway discovered:
2. In times of excess lipid or protein energy sources, certain reactions in the glycolysis pathway may run in reverse in order to produce glucose 6-phosphate which is then used for storage as glycogen or starch.
· Some metabolic pathways flow in a 'cycle' wherein each component of the cycle is a substrate for the subsequent reaction in the cycle, such as in the Krebs Cycle (see below).
· Anabolic and catabolic pathways in eukaryotes often occur independently of each other, separated either physically by compartmentalization within organelles or separated biochemically by the requirement of different enzymes and co-factors.
Several distinct but linked metabolic pathways are used by cells to transfer the energy released by breakdown of fuel molecules into ATPand other small molecules used for energy (e.g. GTP, NADPH, FADH).
These pathways occur within all living organisms in some form:
3. Citric acid cycle / Krebs cycle (not in most obligate anaerobic organisms)
4. Oxidative phosphorylation (not in obligate anaerobic organisms)
Catabolism is characterized by convergence of three major routs toward a final common pathway.
Different proteins, fats and carbohydrates enter the same pathway – tricarboxylic acid cycle.
Anabolism can also be divided into stages, however the anabolic pathways are characterized by divergence.
Monosaccharide synthesis begin with CO2, oxaloacetate, pyruvate or lactate. Amino acids are synthesized from acetyl CoA, pyruvate or keto acids of Krebs cycle. .
Fatty acids are constructed from acetyl CoA.
On the next stage monosaccharides, amino acids and fatty acids are used for the synthesis of polysaccharides, proteins and fats.
Compartmentation of metabolic processes permits:
- separate pools of metabolites within a cell
- simultaneous operation of opposing metabolic paths
- high local concentrations of metabolites
Example: fatty acid synthesis enzymes (cytosol), fatty acid breakdown enzymes (mitochondria).
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.
Glycolysis enzymes are located in the cytosol of cells. Pyruvate enters the mitochondrion to be metabolized further.
Pyruvate dehydrogenase complex is a bridge between glycolysis and aerobic metabolism – citric acid cycle.
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.
Pyruvate freely diffuses through the outer membrane of mitochon-dria through the channels formed by transmembrane proteins porins.
Pyruvate Dehydrogenase catalyzes oxidative decarboxylation of pyruvate, to form acetyl-CoA. The overall reaction is shown below.
Pyruvate dehydrogenase complex is giant, with molecular mass ranging from 4 to 10 million daltons.
Pyruvate Dehydrogenase is a large complex containing many copies of each of three enzymes, E1, E2, and E3.
The inner core of the mammalian Pyruvate Dehydrogenase complex is an icosahedral structure consisting of 60 copies of E2.
At the periphery of the complex are:
· 30 copies of E1 (itself a tetramer with subunits a2b2) and
· 12 copies of E3 (a homodimer), plus 12 copies of an E3 binding protein that links E3 to E2.
Prosthetic groups are listed below
Thiamine pyrophosphate (TPP)
Thiamine pyrophosphate (TPP) is a derivative of thiamine (vitamin B1). Nutritional deficiency of thiamine leads to the disease beriberi. Beriberi affects especially the brain, because TPP is required for carbohydrate metabolism, and the brain depends on glucose metabolism for energy.
A proton readily dissociates from the C that is between N and S in the thiazole ring of TPP. The resulting carbanion (ylid) can attack the electron-deficient keto carbon of pyruvate.
Lipoamide includes a dithiol that undergoes oxidation and reduction.
The carboxyl group at the end of lipoic acid's hydrocarbon chain forms an amide bond to the side-chain amino group of a lysine residue of E2.
A long flexible arm, including hydrocarbon chains of lipoate and the lysine R-group, links the dithiol of each lipoamide to one of two lipoate-binding domains of each E2. Lipoate-binding domains are themselves part of a flexible strand of E2 that extends out from the core of the complex.
The long flexible attachment allows lipoamide functional groups to swing back and forth between E2 active sites in the core of the complex and active sites of E1 & E3 in the outer shell of the complex.
The E3 binding protein (that binds E3 to E2) also has attached lipoamide that can exchange reducing equivalents with lipoamide on E2.
FAD (Flavin Adenine Dinucleotide) is a derivative of the B-vitamin riboflavin (dimethylisoalloxazine-ribitol). The flavin ring system undergoes oxidation/reduction as shown below. Whereas NAD+ is a coenzyme that reversibly binds to enzymes, FAD is a prosthetic group, that is permanently part of the complex.
FAD accepts and donates 2 electrons with 2 protons (2 H):
FAD + 2 e- + 2 H+ �� FADH2
Organic arsenicals are potent inhibitors of lipoamide-containing enzymes such as Pyruvate Dehydrogenase. These highly toxic compounds react with "vicinal" dithiols such as the functional group of lipoamide as shown below.
In the overall reaction, the acetic acid generated is transferred to coenzyme A.
The final electron acceptor is NAD+.
The keto carbon of pyruvate reacts with the carbanion of TPP on E1 to yield an addition compound. The electron-pulling positively charged nitrogen of the thiazole ring promotes loss of CO2. What remains is hydroxyethyl-TPP.
The hydroxyethyl carbanion on TPP of E1 reacts with the disulfide of lipoamide on E2. What was the keto carbon of pyruvate is oxidized to a carboxylic acid, as the disulfide of lipoamide is reduced to a dithiol. The acetate formed by oxidation of the hydroxyethyl moiety is linked to one of the thiols of the reduced lipoamide as a thioester (~).
The acetate is transferred from the thiol of lipoamide to the thiol of coenzyme A, yielding acetyl CoA.
The reduced lipoamide swings over to the E3 active site. Dihydrolipoamide is reoxidized to the disulfide, as 2 e- + 2 H+ are transferred to a disulfide on E3 (disulfide interchange).
The dithiol on E3 is reoxidized as 2 e- + 2 H+ are transferred to FAD. The resulting FADH2 is reoxidized by electron transfer to NAD+, to yield NADH + H+.
Acetyl CoA, a product of the Pyruvate Dehydrogenase reaction, is a central compound in metabolism. The "high energy" thioester linkage makes it an excellent donor of the acetate moiety.
For example, acetyl CoA functions as:
· input to the Krebs Cycle, where the acetate moiety is further degraded to CO2.
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.
Regulation of Pyruvate Dehydrogenase complex.
Pyruvate dehydrogenase is a major regulatory point for entry of materials into the citric acid cycle.. The enzyme is regulated allosterically and by covalent modification.
E2 - inhibited by acetyl-CoA, activated by CoA-SH
E3 - inhibited by NADH, activated by NAD+.
ATP is an allosteric inhibitor of the complex, and AMP is an activator. The activity of this key reaction is coordinated with the energy charge, the [NAD+]/[NADH] ratio, and the ratio of acetylated to free coenzyme A.
Part of the pyruvate dehydrogenase complex, pyruvate dehydrogenase kinase, phosphorylates three specific E1 serine residues, resulting in loss of activity of pyruvate dehydrogenase. NADH and acetyl-CoA both activate the kinase. The serines are dephosphorylated by a specific enzyme called pyruvate dehydrogenase phosphatase that hydrolyzes the phosphates from the E1 subunit of the pyruvate dehydgrogenase complex. This has the effect of activating the complex. The phosphatase is activated by Ca2+and Mg2+. Because ATP and ADP differ in their affinities for Mg2+, the concentration of free Mg2+ reflects the ATP/ADP ratio within the mitochondrion. Thus, pyruvate dehydrogenase responds to ATP levels by being turned off when ATP is abundant and further energy production is unneeded.
In mammalian tissues at rest, much less than half of the total pyruvate dehydrogenase is in the active, nonphosphorylated form. The complex can be turned on when low ATP levels signal a need to generate more ATP. The kinase protein is an integral part of the pyruvate dehydrogenase complex, whereas the phosphatase is but loosely bound.
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.
The Krebs cycle, also known as the tricarboxylic acid cycle (TCA), was first recognized in 1937 by the man for whom it is named, German biochemist Hans Adolph Krebs.
Krebs was educated at the universities of
Krebs is best known for his discovery of the Krebs cycle (or tricarboxylic acid cycle) in 1937. This is a continuation of the work of Carl and Gerty Cori, who had shown howcarbohydrates, such as glycogen, are broken down in the body to lactic acid; Krebs completed the process by working out how the lactic acid is metabolized to carbon dioxideand water. When he began this work little was known apart from the fact that the process involved the consumption of oxygen, which could be increased, according to AlbertSzent-Györgyi, by the four-carbon compounds succinic acid, fumaric acid, malic acid, and oxaloacetic acid. Krebs himself showed in 1937 that the six-carbon citric acid is also involved in the cycle.
By studying the process in pigeon breast muscle Krebs was able to piece together the clues already collected into a coherent scheme. The three-carbon lactic acid is first broken down to a two-carbon molecule unfamiliar to Krebs; it was in fact later identified by Fritz Lipmann as coenzyme A. This then combines with the four-carbon oxaloacetic acid to form the six-carbon citric acid. The citric acid then undergoes a cycle of reactions to be converted to oxaloacetic acid once more. During this cycle two molecules of carbondioxide are given up and hydrogen atoms are released; the hydrogen is then oxidized in the electron transport chain with the production of energy. Much of the detail of this aspect of the cycle was later filled in by Lipmann, with whom Krebs shared the 1953 Nobel Prize for physiology or medicine.
In 1932, with K. Henselheit, Krebs was responsible for the introduction of another cycle. This was the urea cycle, whereby amino acids (the constituents of proteins) eliminate their nitrogen in the form of urea, which is excreted in urine. This left the remainder of the amino acid to give up its potential energy and participate in a variety of metabolic pathways.
Hans A. Krebs, the son of Georg Krebs, an
otolaryngologist, was born in Hildesheim,
In 1935 Krebs went to the
The Ornithine Cycle
To keep organs and tissues alive for biochemical tests, they had been perfused with physiological salines as a substitute for blood. The results were often unsatisfactory. Early in his career Krebs devised the tissue-slice technique. The organ, rapidly removed after the death of the test animal, was cut into thin slices and kept in fresh saline forbiochemical testing. He used this technique in his study of the synthesis of urea by the liver.
It was known that urea is produced in a liver undergoing autolysis, and in 1904 it was shown that the autolysis produces the amino acid arginine, which is acted on catalytically by the enzyme arginase to produce urea. In 1932 Krebs found that, when an amino acid is added to liver, ammonia is liberated and is converted approximately quantitatively into urea. All the amino acids tested gave this result except two. When ornithine was added, the urea production was 10 times the expected amount, and arginine also gave an excess yield of urea. He therefore suggested that ornithine reacted with added ammonia and carbon dioxide to form arginine. Under the action of arginase, the arginine was broken down to urea and ornithine. If ammonia was omitted, there was no appreciable formation of urea. Further, ornithine was not observed to disappear while, with added ammonia, the synthesis of urea was in progress. Krebs therefore concluded that the ornithine acted as a catalyst. Many other substances were tested, but the only one that acted like ornithine was citrulline, and he suggested that citrulline formed a stage midway between ornithine and arginine. His ornithine cycle is still regarded as a sound explanation of the synthesis of urea in the body.
The Citric Acid Cycle
succinic acid→fumaric acid→malic acid→maoxaloacetic acid
He also showed that these reactions were at least in
part catalytic. This was later proved, but the manner of action remained
citric acid→cis -aconitic acid→iso-citric acid→oxalosuccinic acid→alpha-ketoglutamic acid→succinic acid→fumaric acid→malic acid→oxaloacetic acid
Krebs and W. A. Johnson found that citrate was not only rapidly broken down in muscle but was also readily formed provided that oxaloacetate was added. The assumption was that some of the oxaloacetate was broken down to pyruvate or acetate and that the formation of citrate was due to a combination of the remaining oxaloacetate with pyruvate or acetate. But pyruvate or acetate could be derived from carbohydrate. In 1937 Krebs conceived the whole process as a cycle in which an undefined derivative of pyruvate, resulting from the breakdown of carbohydrate, condensed with oxaloacetate to form citric acid. The citric acid then passed through the changes noted above untiloxaloacetic acid was regenerated, and the cycle was repeated. The full cycle is therefore as follows:
citric acid→cis -aconitic acid→iso-citric acid→oxalosuccinic acid→alpha-ketoglutamic acid→succinic acid→fumaric acid→malic acid→oxaloacetic acid+pyruvic acid→citric acid
Since Krebs originally described this cycle, he and others did further work on it. In 1950 Fritz Lipmann showed that the derivative of pyruvic acid that combines with oxaloacetate to form citrate is acetyl-coenzyme A and that this coenzyme is also active at two other points in the cycle. It was shown that acetyl-coenzyme A, in addition to its formation from carbohydrate, is also formed from fatty acids and many amino acids. The Krebs cycle is therefore a most important concept of biochemistry. Krebs shared with Lipmann the Nobel Prize in Physiology or Medicine in 1953.
Among Krebs's other important contributions to biochemistry were his studies of the synthesis of glutamine in brain tissue under the influence of the enzyme glutaminase(1935), the passage of ions across cell membranes (1950), and the effect of primitive intrinsic regulating mechanisms in controlling the metabolism of metazoan cells (1957).
In 1967 Krebs, having reached
Krebs received many honors in addition to his Nobel Prize. In 1947 he was elected a Fellow of the Royal Society, and he was awarded its Royal (1954) and Copley (1961) Medals. He delivered its Croonian Lecture in 1963. He was a member of many foreign scientific societies, and he held honorary doctorates from 14 universities. He received the Gold Medal of the Royal Society of Medicine in 1965, and he was knighted in 1958.
The Krebs cycle refers to a complex series of chemical reactions that produce carbon dioxide and Adenosine triphosphate (ATP), a compound rich in energy. The cycleoccurs by essentially linking two carbon coenzyme with carbon compounds; the created compound then goes through a series of changes that produce energy. This cycle occurs in all cells that utilize oxygen as part of their respiration process; this includes those cells of creatures from the higher animal kingdom such as humans. Carbon dioxide is important for various reasons, the main one being that it stimulates breathing, while ATP provides cells with the energy required for the synthesis of proteins from amino acids and the replication of deoxyribonucleic acid (DNA); both are vital for energy supply and for life to continue. In short, the Krebs cycle constitutes the discovery of the major source of energy in all living organisms.
Within the Krebs cycle, energy in the form of ATP is usually derived from the breakdown of glucose, although fats and proteins can also be utilized as energy sources. Since glucose can pass through cell membranes, it transports energy from one part of the body to another. The Krebs cycle affects all types of life and is, as such, the metabolic pathway within the cells. This pathway chemically converts carbohydrates, fats, and proteins into carbon dioxide, and converts water into serviceable energy.
The Krebs cycle is the second stage of aerobic respiration, the first being glycolysis and last being the electron transport chain; the cycle is a series of stages that every living cell must undergo in order to produce energy. The enzymes that cause each step of the process to occur are all located in the cell's "power plant"; in animals, this power plant is the mitochondria; in plants, it is the chloroplasts; and in microorganisms, it can be found in the cell membrane. The Krebs cycle is also known as the citric acid cycle, because citric acid is the very first product generated by this sequence of chemical conversions, and it is also regenerated at the end of the cycle.
The pyruvate molecules produced during glycolysis contains a lot of energy in the bonds between their molecules. In order to use that energy, the cell must convert it into the form of ATP. To do so, pyruvate molecules are processed through the Kreb Cycle, also known as the citric acid cycle.
(Kerbs Cycle as a drawing)
1. Prior to entering the Krebs Cycle, pyruvate must be converted into acetyl CoA. This is achieved by removing a CO2 molecule from pyruvate and then removing an electron to reduce an NAD+ into NADH. An enzyme called coenzyme A is combined with the remaini ow:
2. Citrate is formed when the acetyl group from acetyl CoA combines with oxaloacetate from the previous Krebs cycle.
3. Citrate is converted into its isomer isocitrate.
4. Isocitrate is oxidized to form the 5-carbon α-ketoglutarate. This step releases one molecule of CO2 and reduces NAD+ to NADH2+.
5. The α-ketoglutarate is oxidized to succinyl CoA, yielding CO2 and NADH2+.
The a-Ketoglutarate Dehydrogenase Complex is
Similar to pyruvate dehydrogenase complex
Same coenzymes, identical mechanisms
E1 - a-ketoglutarate dehydrogenase (with TPP)
E2 – dihydrolipoyl succinyltransferase (with flexible lipoamide prosthetic group)
E3 - dihydrolipoyl dehydrogenase (with FAD)
6. Succinyl CoA releases coenzyme A and phosphorylates ADP into ATP.
In the succinyl CoA synthetase reaction, the thioester bond between HS-CoA and the succinyl group is hydrolyzed.
Since it is a rich in energy bond, the energy released is enough for synthesizing GTP from GDP + (P).
This GTP is equivalent, from the energetic point of view, to ATP. In fact, GTP can transfer the (P) group to ADP to form ATP:
GTP + ADP ————–à GDP + ATP
Since ATP can be produced from this reaction, without participation of the respiratory chain, this process is called Substrate Level Phosphorylation (SLP) in contrast to the Oxidative Phosphorylation (ATP synthesis using the energy released in the Electron Transport Chain).
A few other reactions in metabolism are also coupled with ATP synthesis without participation of the respiratory chain. They are considered also SLP reactions.
7. Succinate is oxidized to fumarate, converting FAD to FADH2.
The Succinate Dehydrogenase Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters, embedded in the inner mitochondrial membrane. Electrons are transferred from succinate to FAD and then to ubiquinone (Q) in electron transport chain. Dehydrogenation is stereospecific; only the trans isomer is formed
8. Fumarate is hydrolized to form malate.
9. Malate is oxidized to oxaloacetate, reducing NAD+ to NADH2+.
We are now back at the beginning of the Krebs Cycle. Because glycolysis produces two pyruvate molecules from one glucose, each glucose is processes through the kreb cycle twice. For each molecule of glucose, six NADH2+, two FADH2, and two ATP.
The sum of all reactions in the citric acid cycle is:
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O → CoA-SH + 3 NADH + 3 H+ + FADH2 + GTP + 2 CO2
(the above reaction is equilibrated if Pi represents the H2PO4- ion, GDP the GDP2- ion and GTP the GTP3- ion).
Two carbons are oxidized to CO2, and the energy from these reactions is stored in GTP, NADH and FADH2. NADH and FADH2 are coenzymes (molecules that enable or enhance enzymes) that store energy and are utilized in oxidative phosphorylation.
Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD+, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced.
The detailed chemical structures have very limited medical significance, but you will find it very much easier to make sense of the other material in this course if you take the trouble to learn them! It may be helpful to follow one particular atom in acetyl CoA all the way round the cycle until it is lost as carbon dioxide, and the coloured boxes are intended to assist this process.
The oxidation of acetyl-CoA to CO2by the TCA cycle is the central process in energy metabolism. However, the TCA cycle also functions in biosynthetic pathways in which intermediates leave the cycle to be converted primarily to glucose, fatty acids, or non-essential amino acids. If TCA cycle anions are removed from the cycle they must be replaced to permit its continued function. This process is termed anaplerosis. Pyruvate carboxylase, which generates oxalacetate directly in the mitochondria, is the major anaplerotic enzyme. Conversely, 4- and 5-carbon intermediates enter the TCA cycle during the catabolism of amino acids. Because the TCA cycle cannot fully oxidize 4- and 5-carbon compounds, these intermediates must be removed from the cycle by a process termed cataplerosis.
Cataplerosis may be linked to biosynthetic processes such as gluconeogenesis in the liver and kidney cortex, fatty acid synthesis in the liver, and glyceroneogenesis in adipose tissue. Cataplerotic enzymes present in many mammalian tissues include P-enolpyruvate carboxykinase (PEPCK), glutamate dehydrogenase, aspartate aminotransferase, and citrate lyase. In this review we have evaluated the roles of anaplerosis and cataplerosis in whole body metabolism.
Biochemical Role of Anaplerosis and Cataplerosis in Function of TCA
The expression anaplerotic sequences was a term used in biochemistry by Sir Hans Kornberg to describe a series of enzymatic reactions or pathways that replenish the pools of metabolic intermediates in the TCA cycle. These intermediates are critical for the functioning of the TCA cycle, the primary role of which is the oxidation of acetyl-CoA to carbon dioxide. The pool of TCA cycle intermediates is sufficient to sustain the oxidative carbon flux over a fairly wide range, so that during high energy consumption (e.g.exercise) or during lower energy consumption (e.g. fasting), there is not a large change in the pool size of TCA intermediates (2). However, in several physiological states, there is a large influx (anaplerosis) of 4- and 5-carbon intermediates into the TCA cycle. Because the TCA cycle cannot act as a carbon sink, anaplerosis must be coupled with the exit of intermediates from the cycle via cataplerosis. The importance of anaplerotic reactions for cellular metabolism is thus apparent. However, the coupling of this process with cataplerosis and the roles that both pathways play in the regulation of amino acid, glucose, and fatty acid metabolism have not been emphasized to a sufficient extent.
The terms anaplerosis and cataplerosis describe reciprocal and correlative reactions involved in the function of the TCA cycle. The enzymatic steps in these processes have long been known, but the overall concept of a linkage between anaplerosis and cataplerosis should be underscored, because the balance between these two processes controls the entry and exit of TCA cycle anions. Anaplerotic and cataplerotic reactions are involved in the ultimate disposal of all metabolic intermediates. The metabolic role of anaplerosis and cataplerosis in amino acid metabolism varies with specific organs and is dependent on the nutritional/metabolic status of the individual. During feeding, the intestine is an important site of catabolism of enterally derived amino acids, whereas in the starved state amino acid catabolism occurs primarily in the kidney, liver, and muscle.
The catabolism of amino acids produces gluconeogenic or ketogenic precursors. The disposal of gluconeogenic anions in the TCA cycle employs anaplerotic and cataplerotic pathways for their terminal oxidation. The only known pathway for the terminal oxidation of leucine is through acetoacetate to acetyl-CoA and subsequent oxidation in the TCA cycle. However, other amino acids also have for their disposal alternate ketogenic pathways for terminal oxidation. Thus, the ketogenic amino acids from proteolysis can be terminally oxidized in muscle, whereas the gluconeogenic amino acids are dependent upon anaplerosis and cataplerosis for conversion to glucose in the liver and kidney before oxidation to CO2 and H2O.
The first reaction of the TCA cycle, citrate synthase, catalyzes the condensation of oxalacetate with acetyl-CoA; the oxalacetate is subsequently regenerated by the reactions of the cycle and condenses with another molecule of acetyl-CoA. However, the TCA cycle also functions in biosynthetic processes in which intermediates are removed from the cycle; this necessitates anaplerotic reactions to replenish TCA cycle intermediates to ensure its continued function. Pyruvate carboxylase, which synthesizes oxalacetate from pyruvate in the mitochondrial matrix, is the archetypical anaplerotic enzyme. The activity of this enzyme is high in many tissues (e.g. 10–12 units/g of liver); acetyl-CoA is a positive allosteric regulator of the enzyme. Anaplerosis is obligatory during both gluconeogenesis and lipogenesis when malate (gluconeogenesis) or citrate (lipogenesis) leaves the mitochondria and is further metabolized to form glucose or fatty acids, respectively.
If intermediates can be added to the TCA cycle, it is equally important to remove them to avoid the accumulation of anions in the mitochondrial matrix. Cataplerosis describes reactions involved in the disposal of TCA cycle intermediates. There are several cataplerotic enzymes; these include PEPCK, aspartate aminotransferase, and glutamate dehydrogenase. Each of these reactions has as substrate a TCA cycle anion that is converted to a product that effectively removes intermediates from the cycle. In the liver and kidney, the role of PEPCK in cataplerosis is of special importance because it is a common route for the generation of PEP from oxalacetate to be used for gluconeogenesis. Alternatively, in muscle, PEP can be converted to pyruvate that can be decarboxylated to acetyl-CoA for subsequent oxidation to CO2 in the TCA cycle.
The regulation of anaplerosis and cataplerosis depends upon the metabolic and physiologic state and the specific tissue/organ involved. For example, during starvation, cataplerosis via phosphoenolpyruvate to support gluconeogenesis may be regulatory in the liver, whereas in the kidney anaplerosis via uptake of glutamine may be regulatory. Anaplerotic and cataplerotic intermediates entering and exiting the TCA cycle are shown below.
Anaplerosis and cataplerosis in the TCA cycle. The TCA cycle is presented with the major anaplerotic and cataplerotic reactions illustrated. These include the net entry of amino acids into the cycle and the generation of oxaloacetate from pyruvate via pyruvate carboxylase. The cataplerotic reactions in the figure illustrate the linkage of this process to both gluconeogenesis and lipogenesis.
The interplay between anaplerotic and cataplerotic reactions in humans was demonstrated by renal metabolism during total, prolonged starvation . Arteriovenous concentration differences of metabolites across the kidneys coupled with urinary nitrogen losses showed that the kidney extracted glutamine and produced urinary ammonium .Concurrently, the kidney released glucose into the blood. It was initially recognized that renal ammoniagenesis was related to ketonuria during prolonged starvation when there is an increase in ketogenesis . However, it was not generally appreciated that the entry (anaplerosis) and removal (cataplerosis) of intermediates into and out of the TCA cycle as related to renal ammoniagenesis and gluconeogenesis had to be balanced. This fundamental principle is poorly understood and is the foundation of this paper.
During prolonged starvation glutamine is transported from muscle to the kidney where the amino and amide groups are used for ammonia formation. The ammonia released from the renal cells serves to titrate the acidity of the tubular urine created by the disassociation of organic acids, primarily β-hydroxybutyric and acetoacetic acids. For ammonia generation to continue, glutamine undergoes anaplerotic reactions to form α-ketoglutarate that enters the TCA cycle and is sequentially converted to malate that leaves the mitochondria. Malate is oxidized in the cytosol to oxalacetate that is subsequently converted to PEP and then to glucose. Thus, anaplerotic and cataplerotic reactions are essential and balanced during renal ammoniagenesis and gluconeogenesis.
The heightened ketonuria that occurs with ketonemia is related to the need for the kidney to generate glucose during total starvation when renal gluconeogenesis accounts for about 50% of the net glucose synthesis . Thus, renal ammoniagenesis and gluconeogenesis are tightly interlocked and dependent upon balanced anaplerotic reactions to replenish the α-ketoglutarate in the TCA cycle and cataplerotic reactions to drain remnant 4-carbon metabolic intermediates from the cycle to synthesize glucose . In addition, there is a metabolic bonus when the kidneys excrete urinary ammonium during starvation. The caloric value of protein is greater when amino acid nitrogen is lost in the urine as ammonium rather than urea because it requires four molecules of ATP to generate a molecule of urea via the urea cycle. In addition, energy is required for the synthesis of creatine and uric acid.
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
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 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
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.