Fundamental concepts of metabolism biochemistry 9788132346494, 8132346491, 1283508079, 9781283508070

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First Edition, 2012

ISBN 978-81-323-4649-4

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Published by: The English Press 4735/22 Prakashdeep Bldg, Ansari Road, Darya Ganj, Delhi - 110002 Email: [email protected] 

Table of Contents Chapter 1 - Metabolism Chapter 2 - Enzyme Chapter 3 - Metabolic Pathway Chapter 4 - Glycolysis Chapter 5 - Citric Acid Cycle Chapter 6 - Oxidative Phosphorylation

Chapter- 1

Metabolism

Structure of the coenzyme adenosine triphosphate, a central intermediate in energy metabolism Metabolism is the set of chemical reactions that happen in living organisms to maintain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories. Catabolism breaks down organic matter, for example to harvest energy in cellular respiration. Anabolism uses energy to construct components of cells such as proteins and nucleic acids. The chemical reactions of metabolism are organized into metabolic pathways, in which one chemical is 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 and will not occur by themselves, by coupling them to spontaneous reactions 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. The metabolism of an organism determines which substances it will find nutritious and which it will find poisonous. For example, some prokaryotes use hydrogen sulfide as a

nutrient, yet this gas is poisonous to animals. The speed of metabolism, the metabolic rate, also influences how much food an organism will require. 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 organisms, being found in species as diverse as the unicellular bacteria Escherichia coli and huge multicellular organisms like elephants. These striking similarities in metabolism are probably due to the high efficiency of these pathways, and their early appearance in evolutionary history.

Key biochemicals

Structure of a triacylglycerol lipid Most of the structures that make up animals, plants and microbes are made from three basic classes of molecule: amino acids, carbohydrates and lipids (often called fats). As these molecules are vital for life, metabolic reactions focus on making these molecules during the construction of cells and tissues, or breaking them down and using them as a source of energy, in the digestion and use of food. Many important biochemicals can be joined together to make polymers such as DNA and proteins. These macromolecules are essential.

Type of molecule

Name of monomer forms

Name of polymer forms Proteins (also called polypeptides)

Amino acids

Amino acids

Carbohydrates

Monosaccharides

Polysaccharides

Nucleic acids

Nucleotides

Polynucleotides

Examples of polymer forms Fibrous proteins and globular proteins Starch, glycogen and cellulose DNA and RNA

Amino acids and proteins Proteins are made of amino acids arranged in a linear chain and joined together by peptide bonds. Many proteins are the enzymes that catalyze the chemical reactions in metabolism. Other proteins have structural or mechanical functions, such as the proteins that form the cytoskeleton, a system of scaffolding that maintains the cell shape. Proteins are also important in cell signaling, immune responses, cell adhesion, active transport across membranes, and the cell cycle.

Lipids Lipids are the most diverse group of biochemicals. Their main structural uses are as part of biological membranes such as the cell membrane, or as a source of energy. Lipids are usually defined as hydrophobic or amphipathic biological molecules that will dissolve in organic solvents such as benzene or chloroform. The fats are a large group of compounds that contain fatty acids and glycerol; a glycerol molecule attached to three fatty acid esters is a triacylglyceride. Several variations on this basic structure exist, including alternate backbones such as sphingosine in the sphingolipids, and hydrophilic groups such as phosphate in phospholipids. Steroids such as cholesterol are another major class of lipids that are made in cells.

Carbohydrates

Glucose can exist in both a straight-chain and ring form

Carbohydrates are straight-chain aldehydes or ketones with many hydroxyl groups that can exist as straight chains or rings. Carbohydrates are the most abundant biological molecules, and fill numerous roles, such as the storage and transport of energy (starch, glycogen) and structural components (cellulose in plants, chitin in animals). The basic carbohydrate units are called monosaccharides and include galactose, fructose, and most importantly glucose. Monosaccharides can be linked together to form polysaccharides in almost limitless ways.

Nucleotides The two nucleic acids, DNA and RNA are polymers of nucleotides, each nucleotide comprising a phosphate group, a ribose sugar group, and a nitrogenous base. Nucleic acids are critical for the storage and use of genetic information, through the processes of transcription and protein biosynthesis. This information is protected by DNA repair mechanisms and propagated through DNA replication. Many viruses have an RNA genome, for example HIV, which uses reverse transcription to create a DNA template from its viral RNA genome. RNA in ribozymes such as spliceosomes and ribosomes is similar to enzymes as it can catalyze chemical reactions. Individual nucleosides are made by attaching a nucleobase to a ribose sugar. These bases are heterocyclic rings containing nitrogen, classified as purines or pyrimidines. Nucleotides also act as coenzymes in metabolic group transfer reactions.

Coenzymes

Structure of the coenzyme acetyl-CoA.The transferable acetyl group is bonded to the sulfur atom at the extreme left. Metabolism involves a vast array of chemical reactions, but most fall under a few basic types of reactions that involve the transfer of functional groups. This common chemistry allows cells to use a small set of metabolic intermediates to carry chemical groups between different reactions. These group-transfer intermediates are called coenzymes. Each class of group-transfer reaction is carried out by a particular coenzyme, which is the substrate for a set of enzymes that produce it, and a set of enzymes that consume it. These coenzymes are therefore continuously being made, consumed and then recycled.

One central coenzyme is adenosine triphosphate (ATP), the universal energy currency of cells. This nucleotide is used to transfer chemical energy between different chemical reactions. There is only a small amount of ATP in cells, but as it is continuously regenerated, the human body can use about its own weight in ATP per day. ATP acts as a bridge between catabolism and anabolism, with catabolic reactions generating ATP and anabolic reactions consuming it. It also serves as a carrier of phosphate groups in phosphorylation reactions. A vitamin is an organic compound needed in small quantities that cannot be made in the cells. In human nutrition, most vitamins function as coenzymes after modification; for example, all water-soluble vitamins are phosphorylated or are coupled to nucleotides when they are used in cells. Nicotinamide adenine dinucleotide (NADH), a derivative of vitamin B3 (niacin), is an important coenzyme that acts as a hydrogen acceptor. Hundreds of separate types of dehydrogenases remove electrons from their substrates and reduce NAD+ into NADH. This reduced form of the coenzyme is then a substrate for any of the reductases in the cell that need to reduce their substrates. Nicotinamide adenine dinucleotide exists in two related forms in the cell, NADH and NADPH. The NAD+/NADH form is more important in catabolic reactions, while NADP+/NADPH is used in anabolic reactions.

Structure of hemoglobin. The protein subunits are in red and blue, and the ironcontaining heme groups in green. From PDB 1GZX.

Minerals and cofactors Inorganic elements play critical roles in metabolism; some are abundant (e.g. sodium and potassium) while others function at minute concentrations. About 99% of mammals' mass are the elements carbon, nitrogen, calcium, sodium, chlorine, potassium, hydrogen, phosphorus, oxygen and sulfur. The organic compounds (proteins, lipids and carbohydrates) contain the majority of the carbon and nitrogen and most of the oxygen and hydrogen is present as water. The abundant inorganic elements act as ionic electrolytes. The most important ions are sodium, potassium, calcium, magnesium, chloride, phosphate, and the organic ion bicarbonate. The maintenance of precise gradients across cell membranes maintains osmotic pressure and pH. Ions are also critical for nerves and muscles, as action potentials in these tissues are produced by the exchange of electrolytes between the extracellular fluid and the cytosol. Electrolytes enter and leave cells through proteins in the cell membrane called ion channels. For example, muscle contraction depends upon the movement of calcium, sodium and potassium through ion channels in the cell membrane and T-tubules. The transition metals are usually present as trace elements in organisms, with zinc and iron being most abundant. These metals are used in some proteins as cofactors and are essential for the activity of enzymes such as catalase and oxygen-carrier proteins such as hemoglobin. These cofactors are bound tightly to a specific protein; although enzyme cofactors can be modified during catalysis, cofactors always return to their original state after catalysis has taken place. The metal micronutrients are taken up into organisms by specific transporters and bound to storage proteins such as ferritin or metallothionein when not being used.

Catabolism Catabolism is the set of metabolic processes that break down large molecules. These include breaking down and oxidizing food molecules. The purpose of the catabolic reactions is to provide the energy and components needed by anabolic reactions. The exact nature of these catabolic reactions differ from organism to organism and organisms can be classified based on their sources of energy and carbon (their primary nutritional groups), as shown in the table below. Organic molecules being used as a source of energy in organotrophs, while lithotrophs use inorganic substrates and phototrophs capture sunlight as chemical energy. However, all these different forms of metabolism depend on redox reactions that involve the transfer of electrons from reduced donor molecules such as organic molecules, water, ammonia, hydrogen sulfide or ferrous ions to acceptor molecules such as oxygen, nitrate or sulfate. In animals these reactions involve complex organic molecules being broken down to simpler molecules, such as carbon dioxide and water. In photosynthetic organisms such as plants and cyanobacteria, these electrontransfer reactions do not release energy, but are used as a way of storing energy absorbed from sunlight.

Classification of organisms based on their metabolism sunlight photoenergy source preformed chemomolecules organic organoelectron compound donor inorganic troph lithocompound organic heterocompound carbon source inorganic autocompound The most common set of catabolic reactions in animals can be separated into three main stages. In the first, large organic molecules such as proteins, polysaccharides or lipids are digested into their smaller components outside cells. Next, these smaller molecules are taken up by cells and converted to yet smaller molecules, usually acetyl coenzyme A (acetyl-CoA), which releases some energy. Finally, 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.

Digestion Macromolecules such as starch, cellulose or proteins cannot be rapidly taken up by cells and need to be broken into their smaller units before they can be used in cell metabolism. Several common classes of enzymes digest these polymers. These digestive enzymes include proteases that digest proteins into amino acids, as well as glycoside hydrolases that digest polysaccharides into monosaccharides. Microbes simply secrete digestive enzymes into their surroundings, while animals only secrete these enzymes from specialized cells in their guts. The amino acids or sugars released by these extracellular enzymes are then pumped into cells by specific active transport proteins.

A simplified outline of the catabolism of proteins, carbohydrates and fats

Energy from organic compounds Carbohydrate catabolism is the breakdown of carbohydrates into smaller units. Carbohydrates are usually taken into cells once they have been digested into monosaccharides. 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 as ribose, 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 by beta 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 (discussed below).

Energy transformations Oxidative phosphorylation

Structure of ATP synthase. The proton channel and rotating stalk are shown in blue and the synthase subunits in red. In oxidative phosphorylation, the electrons removed from food molecules in pathways such as the citric acid cycle are transferred to oxygen and the energy released is used to make ATP. This is done in eukaryotes by a series of proteins in the membranes of

mitochondria called the electron transport chain. In prokaryotes, these proteins are found in the cell's inner membrane. These proteins use the energy released from passing electrons from reduced molecules like NADH onto oxygen to pump protons across a membrane. Pumping protons out of the mitochondria creates a proton concentration difference across the membrane and generates an electrochemical gradient. This force drives protons back into the mitochondrion through the base of an enzyme called ATP synthase. The flow of protons makes the stalk subunit rotate, causing the active site of the synthase domain to change shape and phosphorylate adenosine diphosphate - turning it into ATP.

Energy from inorganic compounds Chemolithotrophy is a type of metabolism found in prokaryotes where energy is obtained from the oxidation of inorganic compounds. These organisms can use hydrogen, reduced sulfur compounds (such as sulfide, hydrogen sulfide and thiosulfate), ferrous iron (FeII) or ammonia as sources of reducing power and they gain energy from the oxidation of these compounds with electron acceptors such as oxygen or nitrite. These microbial processes are important in global biogeochemical cycles such as acetogenesis, nitrification and denitrification and are critical for soil fertility.

Energy from light The energy in sunlight is captured by plants, cyanobacteria, purple bacteria, green sulfur bacteria and some protists. This process is often coupled to the conversion of carbon dioxide into organic compounds, as part of photosynthesis, which is discussed below. The energy capture and carbon fixation systems can however operate separately in prokaryotes, as purple bacteria and green sulfur bacteria can use sunlight as a source of energy, while switching between carbon fixation and the fermentation of organic compounds. In many organisms the capture of solar energy is similar in principle to oxidative phosphorylation, as it involves energy being stored as a proton concentration gradient and this proton motive force then driving ATP synthesis. The electrons needed to drive this electron transport chain come from light-gathering proteins called photosynthetic reaction centres or rhodopsins. Reaction centers are classed into two types depending on the type of photosynthetic pigment present, with most photosynthetic bacteria only having one type, while plants and cyanobacteria have two. In plants, algae, and cyanobacteria, photosystem II uses light energy to remove electrons from water, releasing oxygen as a waste product. The electrons then flow to the cytochrome b6f complex, which uses their energy to pump protons across the thylakoid membrane in the chloroplast. These protons move back through the membrane as they drive the ATP synthase, as before. The electrons then flow through photosystem I and can then either be used to reduce the coenzyme NADP+, for use in the Calvin cycle which is discussed below, or recycled for further ATP generation.

Anabolism 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. 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.

Carbon fixation

Plant cells (bounded by purple walls) filled with chloroplasts (green), which are the site of photosynthesis

Photosynthesis is the synthesis of carbohydrates from sunlight and carbon dioxide (CO2). In plants, cyanobacteria and algae, oxygenic photosynthesis splits water, with oxygen produced as a waste product. This process uses the ATP and NADPH produced by the photosynthetic reaction centres, as described above, to convert CO2 into glycerate 3phosphate, which can then be converted into glucose. This carbon-fixation reaction is carried out by the enzyme RuBisCO as part of the Calvin – Benson cycle. Three types of photosynthesis occur in plants, C3 carbon fixation, C4 carbon fixation and CAM photosynthesis. These differ by the route that carbon dioxide takes to the Calvin cycle, with C3 plants fixing CO2 directly, while C4 and CAM photosynthesis incorporate the CO2 into other compounds first, as adaptations to deal with intense sunlight and dry conditions. In photosynthetic prokaryotes the mechanisms of carbon fixation are more diverse. Here, carbon dioxide can be fixed by the Calvin – Benson cycle, a reversed citric acid cycle, or the carboxylation of acetyl-CoA. Prokaryotic chemoautotrophs also fix CO2 through the Calvin – Benson cycle, but use energy from inorganic compounds to drive the reaction.

Carbohydrates and glycans In carbohydrate anabolism, simple organic acids can be converted into monosaccharides such as glucose and then used to assemble polysaccharides such as starch. The generation of glucose from compounds like pyruvate, lactate, glycerol, glycerate 3-phosphate and amino acids is called gluconeogenesis. Gluconeogenesis converts pyruvate to glucose-6phosphate through a series of intermediates, many of which are shared with glycolysis. However, this pathway is not simply glycolysis run in reverse, as several steps are catalyzed by non-glycolytic enzymes. This is important as it allows the formation and breakdown of glucose to be regulated separately and prevents both pathways from running simultaneously in a futile cycle. Although fat is a common way of storing energy, in vertebrates such as humans the fatty acids in these stores cannot be converted to glucose through gluconeogenesis as these organisms cannot convert acetyl-CoA into pyruvate; plants do, but animals do not, have the necessary enzymatic machinery. As a result, after long-term starvation, vertebrates need to produce ketone bodies from fatty acids to replace glucose in tissues such as the brain that cannot metabolize fatty acids. In other organisms such as plants and bacteria, this metabolic problem is solved using the glyoxylate cycle, which bypasses the decarboxylation step in the citric acid cycle and allows the transformation of acetyl-CoA to oxaloacetate, where it can be used for the production of glucose. Polysaccharides and glycans are made by the sequential addition of monosaccharides by glycosyltransferase from a reactive sugar-phosphate donor such as uridine diphosphate glucose (UDP-glucose) to an acceptor hydroxyl group on the growing polysaccharide. As any of the hydroxyl groups on the ring of the substrate can be acceptors, the polysaccharides produced can have straight or branched structures. The polysaccharides produced can have structural or metabolic functions themselves, or be transferred to lipids and proteins by enzymes called oligosaccharyltransferases.

Fatty acids, isoprenoids and steroids

Simplified version of the steroid synthesis pathway with the intermediates isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP) and squalene shown. Some intermediates are omitted for clarity. Fatty acids are made by fatty acid synthases that polymerize and then reduce acetyl-CoA units. The acyl chains in the fatty acids are extended by a cycle of reactions that add the actyl group, reduce it to an alcohol, dehydrate it to an alkene group and then reduce it again to an alkane group. The enzymes of fatty acid biosynthesis are divided into two groups, in animals and fungi all these fatty acid synthase reactions are carried out by a single multifunctional type I protein, while in plant plastids and bacteria separate type II enzymes perform each step in the pathway.

Terpenes and isoprenoids are a large class of lipids that include the carotenoids and form the largest class of plant natural products. These compounds are made by the assembly and modification of isoprene units donated from the reactive precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate. These precursors can be made in different ways. In animals and archaea, the mevalonate pathway produces these compounds from acetyl-CoA, while in plants and bacteria the non-mevalonate pathway uses pyruvate and glyceraldehyde 3-phosphate as substrates. One important reaction that uses these activated isoprene donors is steroid biosynthesis. Here, the isoprene units are joined together to make squalene and then folded up and formed into a set of rings to make lanosterol. Lanosterol can then be converted into other steroids such as cholesterol and ergosterol.

Proteins Organisms vary in their ability to synthesize the 20 common amino acids. Most bacteria and plants can synthesize all twenty, but mammals can synthesize only eleven nonessential amino acids. Thus, nine essential amino acids must be obtained from food. All amino acids are synthesized from intermediates in glycolysis, the citric acid cycle, or the pentose phosphate pathway. Nitrogen is provided by glutamate and glutamine. Amino acid synthesis depends on the formation of the appropriate alpha-keto acid, which is then transaminated to form an amino acid. Amino acids are made into proteins by being joined together in a chain by peptide bonds. Each different protein has a unique sequence of amino acid residues: this is its primary structure. Just as the letters of the alphabet can be combined to form an almost endless variety of words, amino acids can be linked in varying sequences to form a huge variety of proteins. Proteins are made from amino acids that have been activated by attachment to a transfer RNA molecule through an ester bond. This aminoacyl-tRNA precursor is produced in an ATP-dependent reaction carried out by an aminoacyl tRNA synthetase. This aminoacyl-tRNA is then a substrate for the ribosome, which joins the amino acid onto the elongating protein chain, using the sequence information in a messenger RNA.

Nucleotide synthesis and salvage Nucleotides are made from amino acids, carbon dioxide and formic acid in pathways that require large amounts of metabolic energy. Consequently, most organisms have efficient systems to salvage preformed nucleotides. Purines are synthesized as nucleosides (bases attached to ribose). Both adenine and guanine are made from the precursor nucleoside inosine monophosphate, which is synthesized using atoms from the amino acids glycine, glutamine, and aspartic acid, as well as formate transferred from the coenzyme tetrahydrofolate. Pyrimidines, on the other hand, are synthesized from the base orotate, which is formed from glutamine and aspartate.

Xenobiotics and redox metabolism All organisms are constantly exposed to compounds that they cannot use as foods and would be harmful if they accumulated in cells, as they have no metabolic function. These potentially damaging compounds are called xenobiotics. Xenobiotics such as synthetic drugs, natural poisons and antibiotics are detoxified by a set of xenobiotic-metabolizing enzymes. In humans, these include cytochrome P450 oxidases, UDPglucuronosyltransferases, and glutathione S-transferases. This system of enzymes acts in three stages to firstly oxidize the xenobiotic (phase I) and then conjugate water-soluble groups onto the molecule (phase II). The modified water-soluble xenobiotic can then be pumped out of cells and in multicellular organisms may be further metabolized before being excreted (phase III). In ecology, these reactions are particularly important in microbial biodegradation of pollutants and the bioremediation of contaminated land and oil spills. Many of these microbial reactions are shared with multicellular organisms, but due to the incredible diversity of types of microbes these organisms are able to deal with a far wider range of xenobiotics than multicellular organisms, and can degrade even persistent organic pollutants such as organochloride compounds. A related problem for aerobic organisms is oxidative stress. Here, processes including oxidative phosphorylation and the formation of disulfide bonds during protein folding produce reactive oxygen species such as hydrogen peroxide. These damaging oxidants are removed by antioxidant metabolites such as glutathione and enzymes such as catalases and peroxidases.

Thermodynamics of living organisms Living organisms must obey the laws of thermodynamics, which describe the transfer of heat and work. The second law of thermodynamics states that in any closed system, the amount of entropy (disorder) will tend to increase. Although living organisms' amazing complexity appears to contradict this law, life is possible as all organisms are open systems that exchange matter and energy with their surroundings. Thus living systems are not in equilibrium, but instead are dissipative systems that maintain their state of high complexity by causing a larger increase in the entropy of their environments. The metabolism of a cell achieves this by coupling the spontaneous processes of catabolism to the non-spontaneous processes of anabolism. In thermodynamic terms, metabolism maintains order by creating disorder.

Regulation and control As the environments of most organisms are constantly changing, the reactions of metabolism must be finely regulated to maintain a constant set of conditions within cells, a condition called homeostasis. Metabolic regulation also allows organisms to respond to signals and interact actively with their environments. Two closely linked concepts are important for understanding how metabolic pathways are controlled. Firstly, the regulation of an enzyme in a pathway is how its activity is increased and decreased in

response to signals. Secondly, the control exerted by this enzyme is the effect that these changes in its activity have on the overall rate of the pathway (the flux through the pathway). For example, an enzyme may show large changes in activity (i.e. it is highly regulated) but if these changes have little effect on the flux of a metabolic pathway, then this enzyme is not involved in the control of the pathway.

Effect of insulin on glucose uptake and metabolism. Insulin binds to its receptor (1) which in turn starts many protein activation cascades (2). These include: translocation of Glut-4 transporter to the plasma membrane and influx of glucose (3), glycogen synthesis (4), glycolysis (5) and fatty acid synthesis (6). There are multiple levels of metabolic regulation. In intrinsic regulation, the metabolic pathway self-regulates to respond to changes in the levels of substrates or products; for example, a decrease in the amount of product can increase the flux through the pathway to compensate. This type of regulation often involves allosteric regulation of the activities of multiple enzymes in the pathway. Extrinsic control involves a cell in a multicellular organism changing its metabolism in response to signals from other cells. These signals are usually in the form of soluble messengers such as hormones and growth factors and are detected by specific receptors on the cell surface. These signals are then transmitted inside the cell by second messenger systems that often involved the phosphorylation of proteins. A very well understood example of extrinsic control is the regulation of glucose metabolism by the hormone insulin. Insulin is produced in response to rises in blood glucose levels. Binding of the hormone to insulin receptors on cells then activates a cascade of protein kinases that cause the cells to take up glucose and convert it into storage molecules such as fatty acids and glycogen. The metabolism of glycogen is controlled by activity of phosphorylase, the enzyme that breaks down glycogen, and

glycogen synthase, the enzyme that makes it. These enzymes are regulated in a reciprocal fashion, with phosphorylation inhibiting glycogen synthase, but activating phosphorylase. Insulin causes glycogen synthesis by activating protein phosphatases and producing a decrease in the phosphorylation of these enzymes.

Evolution

Evolutionary tree showing the common ancestry of organisms from all three domains of life. Bacteria are colored blue, eukaryotes red, and archaea green. Relative positions of some of the phyla included are shown around the tree. The central pathways of metabolism described above, such as glycolysis and the citric acid cycle, are present in all three domains of living things and were present in the last universal ancestor. This universal ancestral cell was prokaryotic and probably a methanogen that had extensive amino acid, nucleotide, carbohydrate and lipid metabolism. The retention of these ancient pathways during later evolution may be the result of these reactions being an optimal solution to their particular metabolic problems, with pathways such as glycolysis and the citric acid cycle producing their end products highly efficiently and in a minimal number of steps. The first pathways of enzyme-based metabolism may have been parts of purine nucleotide metabolism, with previous metabolic pathways being part of the ancient RNA world. Many models have been proposed to describe the mechanisms by which novel metabolic pathways evolve. These include the sequential addition of novel enzymes to a short ancestral pathway, the duplication and then divergence of entire pathways as well as the recruitment of pre-existing enzymes and their assembly into a novel reaction pathway. The relative importance of these mechanisms is unclear, but genomic studies have shown

that enzymes in a pathway are likely to have a shared ancestry, suggesting that many pathways have evolved in a step-by-step fashion with novel functions being created from pre-existing steps in the pathway. An alternative model comes from studies that trace the evolution of proteins' structures in metabolic networks, this has suggested that enzymes are pervasively recruited, borrowing enzymes to perform similar functions in different metabolic pathways (evident in the MANET database) These recruitment processes result in an evolutionary enzymatic mosaic. A third possibility is that some parts of metabolism might exist as "modules" that can be reused in different pathways and perform similar functions on different molecules. As well as the evolution of new metabolic pathways, evolution can also cause the loss of metabolic functions. For example, in some parasites metabolic processes that are not essential for survival are lost and preformed amino acids, nucleotides and carbohydrates may instead be scavenged from the host. Similar reduced metabolic capabilities are seen in endosymbiotic organisms.

Investigation and manipulation

Metabolic network of the Arabidopsis thaliana citric acid cycle. Enzymes and metabolites are shown as red squares and the interactions between them as black lines.

Classically, metabolism is studied by a reductionist approach that focuses on a single metabolic pathway. Particularly valuable is the use of radioactive tracers at the wholeorganism, tissue and cellular levels, which define the paths from precursors to final products by identifying radioactively labelled intermediates and products. The enzymes that catalyze these chemical reactions can then be purified and their kinetics and responses to inhibitors investigated. A parallel approach is to identify the small molecules in a cell or tissue; the complete set of these molecules is called the metabolome. Overall, these studies give a good view of the structure and function of simple metabolic pathways, but are inadequate when applied to more complex systems such as the metabolism of a complete cell. An idea of the complexity of the metabolic networks in cells that contain thousands of different enzymes is given by the figure showing the interactions between just 43 proteins and 40 metabolites to the right: the sequences of genomes provide lists containing anything up to 45,000 genes. However, it is now possible to use this genomic data to reconstruct complete networks of biochemical reactions and produce more holistic mathematical models that may explain and predict their behavior. These models are especially powerful when used to integrate the pathway and metabolite data obtained through classical methods with data on gene expression from proteomic and DNA microarray studies. Using these techniques, a model of human metabolism has now been produced, which will guide future drug discovery and biochemical research. These models are now being used in network analysis, to classify human diseases into groups that share common proteins or metabolites. Bacterial metabolic networks seem to be a striking example of bow-tie organization, an architecture able to input a wide range of nutrients and produce a large variety of products and complex macromolecules using a relatively few intermediate common currencies. A major technological application of this information is metabolic engineering. Here, organisms such as yeast, plants or bacteria are genetically modified to make them more useful in biotechnology and aid the production of drugs such as antibiotics or industrial chemicals such as 1,3-propanediol and shikimic acid. These genetic modifications usually aim to reduce the amount of energy used to produce the product, increase yields and reduce the production of wastes.

History

Santorio Santorio in his steelyard balance, from Ars de statica medicina, first published 1614 The term metabolism is derived from the Greek Μεταβολισμός – "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 Santorio in 1614 in his book Ars de statica medicina. He described how he weighed himself before and after eating, sleep, working, sex, fasting, drinking, and excreting. He found that most of the food he took in was lost through what he called "insensible perspiration". 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, 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 was Hans 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 and molecular dynamics simulations. These techniques have allowed the discovery and detailed analysis of the many molecules and metabolic pathways in cells.

Chapter- 2

Enzyme

Human glyoxalase I. Two zinc ions that are needed for the enzyme to catalyze its reaction are shown as purple spheres, and an enzyme inhibitor called S-hexylglutathione is shown as a space-filling model, filling the two active sites. Enzymes are proteins that catalyze (i.e., increase or decrease the rates of) chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and they are converted into different molecules, called the products. Almost all processes in a biological cell need enzymes to occur at significant rates. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell. Like all catalysts, enzymes work by lowering the activation energy (Ea‡) for a reaction, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. Enzymes are known to catalyze about 4,000 biochemical reactions. A few RNA molecules called ribozymes also catalyze reactions,

with an important example being some parts of the ribosome. Synthetic molecules called artificial enzymes also display enzyme-like catalysis. Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, chemical environment (e.g., pH), and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins into smaller molecules, making the meat easier to chew).

Etymology and history

Eduard Buchner As early as the late 18th and early 19th centuries, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified. In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms. 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."

In 1877, German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which comes from Greek ενζυμον, "in leaven", to describe this process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment was used to refer to chemical activity produced by living organisms. In 1897, Eduard Buchner submitted his first paper on the ability of yeast extracts that lacked any living yeast cells to ferment sugar. In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast cells in the mixture. He named the enzyme that brought about the fermentation of sucrose "zymase". In 1907, he received the Nobel Prize in Chemistry "for his biochemical research and his discovery of cell-free fermentation". Following Buchner's example, enzymes are usually named according to the reaction they carry out. Typically, to generate the name of an enzyme, the suffix -ase is added to the name of its substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA polymerase forms DNA polymers). Having shown that enzymes could function outside a living cell, the next step was to determine their biochemical nature. Many early workers noted that enzymatic activity was associated with proteins, but several scientists (such as Nobel laureate Richard Willstätter) argued that proteins were merely carriers for the true enzymes and that proteins per se were incapable of catalysis. However, in 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it; Sumner did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively proved by Northrop and Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel Prize in Chemistry. This discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography. This was first done for lysozyme, an enzyme found in tears, saliva and egg whites that digests the coating of some bacteria; the structure was solved by a group led by David Chilton Phillips and published in 1965. This highresolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail.

Structures and mechanisms

Ribbon diagram showing human carbonic anhydrase II. The grey sphere is the zinc cofactor in the active site. Diagram drawn from PDB 1MOO. Enzymes are generally globular proteins and range from just 62 amino acid residues in size, for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty acid synthase. A small number of RNA-based biological catalysts exist, with the most common being the ribosome; these are referred to as either RNA-enzymes or ribozymes. The activities of enzymes are determined by their three-dimensional structure. However, although structure does determine function, predicting a novel enzyme's activity just from its structure is a very difficult problem that has not yet been solved. Most enzymes are much larger than the substrates they act on, and only a small portion of the enzyme (around 3–4 amino acids) is directly involved in catalysis. The region that contains these catalytic residues, binds the substrate, and then carries out the reaction is

known as the active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis. Some enzymes also have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction catalyzed. This binding can serve to increase or decrease the enzyme's activity, providing a means for feedback regulation. Like all proteins, enzymes are long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a specific structure, which has unique properties. Individual protein chains may sometimes group together to form a protein complex. Most enzymes can be denatured—that is, unfolded and inactivated—by heating or chemical denaturants, which disrupt the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible. Structures of enzymes in complex with substrates or substrate analogs during a reaction may be obtained using Time resolved crystallography methods.

Specificity Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity. Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases. Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes. Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of different substrates. It has been suggested that this broad substrate specificity is important for the evolution of new biosynthetic pathways.

"Lock and key" model Enzymes are very specific, and it was suggested by the Nobel laureate organic chemist Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.

Diagrams to show the induced fit hypothesis of enzyme action In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site; the amino acid side chains which make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site. The active site continues to change until the substrate is completely bound, at which point the final shape and charge is determined. Induced fit may enhance the fidelity of molecular recognition in the presence of competition and noise via the conformational proofreading mechanism.

Mechanisms Enzymes can act in several ways, all of which lower ΔG‡: •

• • • •

Lowering the activation energy by creating an environment in which the transition state is stabilized (e.g. straining the shape of a substrate—by binding the transition-state conformation of the substrate/product molecules, the enzyme distorts the bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the transition). Lowering the energy of the transition state, but without distorting the substrate, by creating an environment with the opposite charge distribution to that of the transition state. Providing an alternative pathway. For example, temporarily reacting with the substrate to form an intermediate ES complex, which would be impossible in the absence of the enzyme. Reducing the reaction entropy change by bringing substrates together in the correct orientation to react. Considering ΔH‡ alone overlooks this effect. Increases in temperatures speed up reactions. Thus, temperature increases help the enzyme function and develop the end product even faster. However, if heated too

much, the enzyme’s shape deteriorates and the enzyme becomes denatured. Some enzymes like thermolabile enzymes work best at low temperatures. Interestingly, this entropic effect involves destabilization of the ground state, and its contribution to catalysis is relatively small.

Transition State Stabilization The understanding of the origin of the reduction of ΔG‡ requires one to find out how the enzymes can stabilize its transition state more than the transition state of the uncatalyzed reaction. Apparently, the most effective way for reaching large stabilization is the use of electrostatic effects, in particular, by having a relatively fixed polar environment that is oriented toward the charge distribution of the transition state. Such an environment does not exist in the uncatalyzed reaction in water.

Dynamics and function The internal dynamics of enzymes is linked to their mechanism of catalysis. Internal dynamics are the movement of parts of the enzyme's structure, such as individual amino acid residues, a group of amino acids, or even an entire protein domain. These movements occur at various time-scales ranging from femtoseconds to seconds. Networks of protein residues throughout an enzyme's structure can contribute to catalysis through dynamic motions. Protein motions are vital to many enzymes, but whether small and fast vibrations, or larger and slower conformational movements are more important depends on the type of reaction involved. However, although these movements are important in binding and releasing substrates and products, it is not clear if protein movements help to accelerate the chemical steps in enzymatic reactions. These new insights also have implications in understanding allosteric effects and developing new drugs.

Allosteric modulation

Allosteric transition of an enzyme between R and T states, stabilized by an agonist, an inhibitor and a substrate (the MWC model) Allosteric sites are sites on the enzyme that bind to molecules in the cellular environment. The sites form weak, noncovalent bonds with these molecules, causing a change in the conformation of the enzyme. This change in conformation translates to the active site, which then affects the reaction rate of the enzyme. Allosteric interactions can both inhibit and activate enzymes and are a common way that enzymes are controlled in the body.

Cofactors and coenzymes Cofactors Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules called cofactors to be bound for activity. Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds (e.g., flavin and heme). Organic cofactors can be either prosthetic groups, which are tightly bound to an enzyme, or coenzymes, which are released from the enzyme's active site during the reaction. Coenzymes include NADH, NADPH and adenosine triphosphate. These molecules transfer chemical groups between enzymes. An example of an enzyme that contains a cofactor is carbonic anhydrase, and is shown in the ribbon diagram above with a zinc cofactor bound as part of its active site. These tightly bound molecules are usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions.

Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins. An apoenzyme together with its cofactor(s) is called a holoenzyme (this is the active form). Most cofactors are not covalently attached to an enzyme, but are very tightly bound. However, organic prosthetic groups can be covalently bound (e.g., thiamine pyrophosphate in the enzyme pyruvate dehydrogenase). The term "holoenzyme" can also be applied to enzymes that contain multiple protein subunits, such as the DNA polymerases; here the holoenzyme is the complete complex containing all the subunits needed for activity.

Coenzymes

Space-filling model of the coenzyme NADH Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Tightly bound coenzymes can be called allosteric groups. Coenzymes are transport chemical groups from one enzyme to another. Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins (compounds which cannot be synthesized

by the body and must be acquired from the diet). The chemical groups carried include the hydride ion (H-) carried by NAD or NADP+, the phosphate group carried by adenosine triphosphate, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine. Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 700 enzymes are known to use the coenzyme NADH. Coenzymes are usually continuously regenerated and their concentrations maintained at a steady level inside the cell: for example, NADPH is regenerated through the pentose phosphate pathway and S-adenosylmethionine by methionine adenosyltransferase. This continuous regeneration means that even small amounts of coenzymes are used very intensively. For example, the human body turns over its own weight in ATP each day.

Thermodynamics

The energies of the stages of a chemical reaction. Substrates need a lot of energy to reach a transition state, which then decays into products. The enzyme stabilizes the transition state, reducing the energy needed to form products.

As all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. Usually, in the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly. However, in the absence of the enzyme, other possible uncatalyzed, "spontaneous" reactions might lead to different products, because in those conditions this different product is formed faster. Furthermore, enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavorable one. For example, the hydrolysis of ATP is often used to drive other chemical reactions. Enzymes catalyze the forward and backward reactions equally. They do not alter the equilibrium itself, but only the speed at which it is reached. For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants. (in tissues; high CO2 concentration) (in lungs; low CO2 concentration) Nevertheless, if the equilibrium is greatly displaced in one direction, that is, in a very exergonic reaction, the reaction is effectively irreversible. Under these conditions the enzyme will, in fact, only catalyze the reaction in the thermodynamically allowed direction.

Kinetics

Mechanism for a single substrate enzyme catalyzed reaction. The enzyme (E) binds a substrate (S) and produces a product (P). Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are obtained from enzyme assays. In 1902 Victor Henri proposed a quantitative theory of enzyme kinetics, but his experimental data were not useful because the significance of the hydrogen ion

concentration was not yet appreciated. After Peter Lauritz Sørensen had defined the logarithmic pH-scale and introduced the concept of buffering in 1909 the German chemist Leonor Michaelis and his Canadian postdoc Maud Leonora Menten repeated Henri's experiments and confirmed his equation which is referred to as Henri-MichaelisMenten kinetics (sometimes also Michaelis-Menten kinetics). Their work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today. The major contribution of Henri was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis complex. The enzyme then catalyzes the chemical step in the reaction and releases the product.

Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v). Enzymes can catalyze up to several million reactions per second. For example, the uncatalyzed decarboxylation of orotidine 5'-monophosphate has a half life of 78 million years. However, when the enzyme orotidine 5'-phosphate decarboxylase is added, the same process takes just 25 milliseconds. Enzyme rates depend on solution conditions and substrate concentration. Conditions that denature the protein abolish enzyme activity, such as high temperatures, extremes of pH or high salt concentrations, while raising substrate concentration tends to increase activity. To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of

product formation is seen. This is shown in the saturation curve on the right. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the maximum velocity (Vmax) of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme. However, Vmax is only one kinetic constant of enzymes. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis-Menten constant (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum velocity. Each enzyme has a characteristic Km for a given substrate, and this can show how tight the binding of the substrate is to the enzyme. Another useful constant is kcat, which is the number of substrate molecules handled by one active site per second. The efficiency of an enzyme can be expressed in terms of kcat/Km. This is also called the specificity constant and incorporates the rate constants for all steps in the reaction. Because the specificity constant reflects both affinity and catalytic ability, it is useful for comparing different enzymes against each other, or the same enzyme with different substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is about 108 to 109 (M−1 s−1). At this point every collision of the enzyme with its substrate will result in catalysis, and the rate of product formation is not limited by the reaction rate but by the diffusion rate. Enzymes with this property are called catalytically perfect or kinetically perfect. Example of such enzymes are triose-phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, β-lactamase, and superoxide dismutase. Michaelis-Menten kinetics relies on the law of mass action, which is derived from the assumptions of free diffusion and thermodynamically driven random collision. However, many biochemical or cellular processes deviate significantly from these conditions, because of macromolecular crowding, phase-separation of the enzyme/substrate/product, or one or two-dimensional molecular movement. In these situations, a fractal MichaelisMenten kinetics may be applied. Some enzymes operate with kinetics which are faster than diffusion rates, which would seem to be impossible. Several mechanisms have been invoked to explain this phenomenon. Some proteins are believed to accelerate catalysis by drawing their substrate in and pre-orienting them by using dipolar electric fields. Other models invoke a quantum-mechanical tunneling explanation, whereby a proton or an electron can tunnel through activation barriers, although for proton tunneling this model remains somewhat controversial. Quantum tunneling for protons has been observed in tryptamine. This suggests that enzyme catalysis may be more accurately characterized as "through the barrier" rather than the traditional model, which requires substrates to go "over" a lowered energy barrier.

Inhibition

Competitive inhibitors bind reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor. Substrate and inhibitor compete for the enzyme.

Types of inhibition. This classification was introduced by W.W. Cleland Enzyme reaction rates can be decreased by various types of enzyme inhibitors. Competitive inhibition In competitive inhibition, the inhibitor and substrate compete for the enzyme (i.e., they can not bind at the same time). Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of folic acid and this drug are shown in the figure to the right bottom. Note that binding of the inhibitor need not be to the substrate binding site (as frequently stated), if binding of the inhibitor changes the

conformation of the enzyme to prevent substrate binding and vice versa. In competitive inhibition the maximal velocity of the reaction is not changed, but higher substrate concentrations are required to reach a given velocity, increasing the apparent Km. Uncompetitive inhibition In uncompetitive inhibition the inhibitor can not bind to the free enzyme, but only to the ES-complex. The EIS-complex thus formed is enzymatically inactive. This type of inhibition is rare, but may occur in multimeric enzymes. Non-competitive inhibition Non-competitive inhibitors can bind to the enzyme at the binding site at the same time as the substrate,but not to the active site. Both the EI and EIS complexes are enzymatically inactive. Because the inhibitor can not be driven from the enzyme by higher substrate concentration (in contrast to competitive inhibition), the apparent Vmax changes. But because the substrate can still bind to the enzyme, the Km stays the same. Mixed inhibition This type of inhibition resembles the non-competitive, except that the EIS-complex has residual enzymatic activity.This type of inhibitor does not follow Michaelis-Menten equation. In many organisms inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one substance in the organism, that substance may act as an inhibitor for the enzyme at the beginning of the pathway that produces it, causing production of the substance to slow down or stop when there is sufficient amount. This is a form of negative feedback. Enzymes which are subject to this form of regulation are often multimeric and have allosteric binding sites for regulatory substances. Their substrate/velocity plots are not hyperbolar, but sigmoidal (S-shaped).

The coenzyme folic acid (left) and the anti-cancer drug methotrexate (right) are very similar in structure. As a result, methotrexate is a competitive inhibitor of many enzymes that use folates.

Irreversible inhibitors react with the enzyme and form a covalent adduct with the protein. The inactivation is irreversible. These compounds include eflornithine a drug used to treat the parasitic disease sleeping sickness. Penicillin and Aspirin also act in this manner. With these drugs, the compound is bound in the active site and the enzyme then converts the inhibitor into an activated form that reacts irreversibly with one or more amino acid residues. Uses of inhibitors Since inhibitors modulate the function of enzymes they are often used as drugs. An common example of an inhibitor that is used as a drug is aspirin, which inhibits the COX1 and COX-2 enzymes that produce the inflammation messenger prostaglandin, thus suppressing pain and inflammation. However, other enzyme inhibitors are poisons. For example, the poison cyanide is an irreversible enzyme inhibitor that combines with the copper and iron in the active site of the enzyme cytochrome c oxidase and blocks cellular respiration.

Biological function Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases. They also generate movement, with myosin hydrolysing ATP to generate muscle contraction and also moving cargo around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps involved in active transport. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies. Viruses can also contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase. An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyse the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants which have herbivorous diets, microorganisms in the gut produce another enzyme, cellulase to break down the cellulose cell walls of plant fiber.

Glycolytic enzymes and their functions in the metabolic pathway of glycolysis Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyze the same reaction in parallel, this can allow more complex regulation: with for example a low constant activity being provided by one enzyme but an inducible high activity from a second enzyme. Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps, nor be fast enough to serve the needs of the cell. Indeed, a metabolic pathway such as glycolysis could not exist independently of enzymes. Glucose, for example, can react directly with ATP to become phosphorylated at one or more of its carbons. In the absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is added, these slow reactions continue to take place except that phosphorylation at carbon 6 occurs so rapidly that if the mixture is tested a short time later, glucose-6-phosphate is found to be the only significant product. Consequently, the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.

Control of activity There are five main ways that enzyme activity is controlled in the cell. 1. Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell in response to changes in the cell's environment. This form of gene regulation is called enzyme induction and inhibition. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam

2.

3.

4.

5.

ring within the penicillin molecule. Another example are enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause drug interactions. Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic reticulum and the Golgi apparatus and used by a different set of enzymes as a source of energy in the mitochondrion, through β-oxidation. Enzymes can be regulated by inhibitors and activators. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps allocate materials and energy economically, and prevents the manufacture of excess end products. The control of enzymatic action helps to maintain a stable internal environment in living organisms. Enzymes can be regulated through post-translational modification. This can include phosphorylation, myristoylation and glycosylation. For example, in the response to insulin, the phosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or degradation of glycogen and allows the cell to respond to changes in blood sugar. Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. This type of inactive precursor to an enzyme is known as a zymogen. Some enzymes may become activated when localized to a different environment (e.g. from a reducing (cytoplasm) to an oxidizing (periplasm) environment, high pH to low pH etc.). For example, hemagglutinin in the influenza virus is activated by a conformational change caused by the acidic conditions, these occur when it is taken up inside its host cell and enters the lysosome.

Involvement in disease

Phenylalanine hydroxylase. Created from PDB 1KW0 Since the tight control of enzyme activity is essential for homeostasis, any malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to a genetic disease. The importance of enzymes is shown by the fact that a lethal illness can be caused by the malfunction of just one type of enzyme out of the thousands of types present in our bodies. One example is the most common type of phenylketonuria. A mutation of a single amino acid in the enzyme phenylalanine hydroxylase, which catalyzes the first step in the degradation of phenylalanine, results in build-up of phenylalanine and related products. This can lead to mental retardation if the disease is untreated.

Another example is when germline mutations in genes coding for DNA repair enzymes cause hereditary cancer syndromes such as xeroderma pigmentosum. Defects in these enzymes cause cancer since the body is less able to repair mutations in the genome. This causes a slow accumulation of mutations and results in the development of many types of cancer in the sufferer.

Naming conventions An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase. Examples are lactase, alcohol dehydrogenase and DNA polymerase. This may result in different enzymes, called isozymes, with the same function having the same basic name. Isoenzymes have a different amino acid sequence and might be distinguished by their optimal pH, kinetic properties or immunologically. Isoenzyme and isozyme are homologous proteins. Furthermore, the normal physiological reaction an enzyme catalyzes may not be the same as under artificial conditions. This can result in the same enzyme being identified with two different names. E.g. Glucose isomerase, used industrially to convert glucose into the sweetener fructose, is a xylose isomerase in vivo. The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers; each enzyme is described by a sequence of four numbers preceded by "EC". The first number broadly classifies the enzyme based on its mechanism. The top-level classification is • • • • • •

EC 1 Oxidoreductases: catalyze oxidation/reduction reactions EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group) EC 3 Hydrolases: catalyze the hydrolysis of various bonds EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation EC 5 Isomerases: catalyze isomerization changes within a single molecule EC 6 Ligases: join two molecules with covalent bonds.

According to the naming conventions, enzymes are generally classified into six main family classes and many sub-family classes. Some web-servers, e.g., EzyPred and bioinformatics tools have been developed to predict which main family class and subfamily class an enzyme molecule belongs to according to its sequence information alone via the pseudo amino acid composition.

Industrial applications Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. However, enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures. Consequently, protein engineering is an active

area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. These efforts have begun to be successful, and a few enzymes have now been desiged "from scratch" to catalyze reactions that do not occur in nature. Application

Enzymes used

Uses

Production of sugars from starch, such as in making high-fructose corn syrup. Food processing In baking, catalyze Amylases from fungi and breakdown of starch in the plants flour to sugar. Yeast fermentation of sugar produces the carbon dioxide that raises the Amylases catalyze the release of dough. simple sugars from starch. Biscuit manufacturers use Proteases them to lower the protein level of flour. Baby foods

Trypsin

To predigest baby foods

Enzymes from barley are released during the mashing stage of beer production.

They degrade starch and proteins to produce simple sugar, amino acids and peptides that are used by yeast for fermentation.

Industrially produced barley enzymes

Widely used in the brewing process to substitute for the natural enzymes found in barley.

Amylase, glucanases, proteases

Split polysaccharides and proteins in the malt.

Betaglucanases and arabinoxylanases

Improve the wort and beer filtration characteristics.

Brewing industry

Germinating barley used for malt Amyloglucosidase and pullulanases

Low-calorie beer and adjustment of fermentability.

Remove cloudiness produced during storage of beers. Increases fermentation Acetolactatedecarboxylase efficiency by reducing (ALDC) diacetyl formation. Proteases

Fruit juices

Cellulases, pectinases

Clarify fruit juices.

Dairy industry

Rennin, derived from the stomachs of young ruminant animals (like calves and lambs)

Manufacture of cheese, used to hydrolyze protein

Microbially produced enzyme

Now finding increasing use in the dairy industry

Lipases

Is implemented during the production of Roquefort cheese to enhance the ripening of the blue-mould cheese.

Lactases

Break down lactose to glucose and galactose.

Papain

To soften meat for cooking

Amylases, amyloglucosideases and glucoamylases

Converts starch into glucose and various syrups.

Glucose isomerase

Converts glucose into fructose in production of high fructose syrups from starchy materials. These syrups have enhanced sweetening properties and lower calorific values than sucrose for the same level of sweetness.

Amylases, Xylanases, Cellulases and ligninases

Degrade starch to lower viscosity, aiding sizing and coating paper. Xylanases reduce bleach required for decolorising; cellulases smooth fibers, enhance water drainage, and promote ink removal; lipases reduce pitch and lignin-degrading enzymes remove lignin to soften paper.

Cellulases

Used to break down cellulose into sugars that can be fermented

Roquefort cheese

Meat tenderizers

Starch industry

Glucose

Fructose

Paper industry

A paper mill in South Carolina

Biofuel industry

Ligninases

Use of lignin waste

Primarily proteases, produced in an extracellular form from bacteria

Used for presoak conditions and direct liquid applications helping with removal of protein stains from clothes

Amylases

Detergents for machine dish washing to remove resistant starch residues

Lipases

Used to assist in the removal of fatty and oily stains

Cellulases

Used in biological fabric conditioners

Contact lens cleaners

Proteases

To remove proteins on contact lens to prevent infections

Rubber industry

Catalase

To generate oxygen from peroxide to convert latex into foam rubber

Protease (ficin)

Dissolve gelatin off scrap film, allowing recovery of its silver content.

Cellulose in 3D

Biological detergent

Photographic industry

Molecular biology

Part of the DNA double helix

Used to manipulate DNA in genetic engineering, important in pharmacology, agriculture Restriction enzymes, DNA and medicine. Essential ligase and polymerases for restriction digestion and the polymerase chain reaction. Molecular biology is also important in forensic science.

Chapter- 3

Metabolic Pathway

In biochemistry, metabolic pathways are series of chemical reactions occurring within a cell. In each pathway, a principal chemical is modified by a series of chemical reactions. Enzymes catalyze these reactions, and often require dietary minerals, vitamins, and other cofactors in order to function properly. Because of the many chemicals (a.k.a. "metabolites") that may be involved, metabolic pathways can be quite elaborate. In addition, numerous distinct pathways co-exist within a cell. This collection of pathways is called the metabolic network. Pathways are important to the maintenance of homeostasis within an organism. Catabolic (break-down) and Anabolic (synthesis) pathways often work interdependently to create new biomolecules as the final endproducts. A metabolic pathway involves the step-by-step modification of an initial molecule to form another product. The resulting product can be used in one of three ways: • • •

To be used immediately, as the end-product of a metabolic pathway To initiate another metabolic pathway, called a flux generating step To be stored by the cell

A molecule called a substrate enters a metabolic pathway depending on the needs of the cell and the availability of the substrate. An increase in concentration of anabolic and catabolic intermediates and/or end-products may influence the metabolic rate for that particular pathway.

Overview 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:

1. As glucose enters a cell, it is immediately phosphorylated by ATP to glucose 6-phosphate in the irreversible first step. 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 6phosphate which is then used for storage as glycogen or starch. • • •

Metabolic pathways are often regulated by feedback inhibition. 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.

Major metabolic pathways

Cellular respiration

Cellular respiration in a typical eukaryotic cell Cellular respiration (also known as oxidative metabolism) is the set of the metabolic reactions and processes that take place in the cells of organisms to convert biochemical energy from nutrients into adenosine triphosphate (ATP), and then release waste products. The reactions involved in respiration are catabolic reactions that involve the oxidation of one molecule and the reduction of another. Respiration is one of the key ways a cell gains useful energy to fuel cellular reformations. Nutrients commonly used by animal and plant cells in respiration include glucose, amino acids and fatty acids, and a common oxidizing agent (electron acceptor) is molecular oxygen (O2). Bacteria and archaea can also be lithotrophs and these organisms may respire using a broad range of inorganic molecules as electron donors and acceptors, such as sulfur, metal ions, methane or hydrogen. Organisms that use oxygen as a final electron acceptor in respiration are described as aerobic, while those that do not are referred to as anaerobic.

The energy released in respiration is used to synthesize ATP to store this energy. The energy stored in ATP can then be used to drive processes requiring energy, including biosynthesis, locomotion or transportation of molecules across cell membranes.

Aerobic respiration

Aerobic respiration (red arrows) is the main means by which both plants and animals utilize energy in the form of organic compounds that was previously created through photosynthesis (green arrow). Aerobic respiration requires oxygen in order to generate energy (ATP). Although carbohydrates, fats, and proteins can all be processed and consumed as reactant, it is the preferred method of pyruvate breakdown in glycolysis and requires that pyruvate enter the mitochondrion in order to be fully oxidized by the Krebs cycle. The product of this

process is energy in the form of ATP (Adenosine triphosphate), by substrate-level phosphorylation, NADH and FADH2

Simplified reaction:

C6H12O6 (aq) + 6 O2 (g) → 6 CO2 (g) + 6 H2O (l) ΔG = -2880 kJ per mole of C6H12O6

The negative ΔG indicates that the reaction can happen spontaneously The reducing potential of NADH and FADH2 is converted to more ATP through an electron transport chain with oxygen as the "terminal electron acceptor". Most of the ATP produced by aerobic cellular respiration is made by oxidative phosphorylation. This works by the energy released in the consumption of pyruvate being used to create a chemiosmotic potential by pumping protons across a membrane. This potential is then used to drive ATP synthase and produce ATP from ADP. Biology textbooks often state that 38 ATP molecules can be made per oxidised glucose molecule during cellular respiration (2 from glycolysis, 2 from the Krebs cycle, and about 34 from the electron transport system). However, this maximum yield is never quite reached due to losses (leaky membranes) as well as the cost of moving pyruvate and ADP into the mitochondrial matrix and current estimates range around 29 to 30 ATP per glucose. Aerobic metabolism is 19 times more efficient than anaerobic metabolism (which yields 2 mol ATP per 1 mol glucose). They share the initial pathway of glycolysis but aerobic metabolism continues with the Krebs cycle and oxidative phosphorylation. The post glycolytic reactions take place in the mitochondria in eukaryotic cells, and in the cytoplasm in prokaryotic cells.

Oxidative phosphorylation In eukaryotes, oxidative phosphorylation occurs in the mitochondrial cristae. It comprises the electron transport chain that establishes a proton gradient (chemiosmotic potential) across the inner membrane by oxidizing the NADH produced from the Krebs cycle. ATP is synthesised by the ATP synthase enzyme when the chemiosmotic gradient is used to drive the phosphorylation of ADP. The electrons are finally transferred to exogenous oxygen and, with the addition of two protons, water is formed. the table below are for one glucose molecule being fully oxidized into carbon dioxide. It is assumed that all the reduced coenzymes are oxidized by the electron transport chain and used for oxidative phosphorylation.

Step

coenzyme yield

ATP yield

Source of ATP

Glycolysis preparatory phase

-2

Glycolysis pay-off phase

2 NADH

4 6

Phosphorylation of glucose and fructose 6phosphate uses two ATP from the cytoplasm. Substrate-level phosphorylation Oxidative phosphorylation

2 NADH

6

Oxidative phosphorylation

6 NADH 2 FADH2

2 18 4

Substrate-level phosphorylation Oxidative phosphorylation Oxidative phosphorylation

Oxidative decarboxylation of pyruvate Krebs cycle

Total yield

From the complete oxidation of one glucose 38 ATP molecule to carbon dioxide and oxidation of all the reduced coenzymes.

Although there is a theoretical yield of 38 ATP molecules per glucose during cellular respiration, such conditions are generally not realized due to losses such as the cost of moving pyruvate (from glycolysis), phosphate, and ADP (substrates for ATP synthesis) into the mitochondria. All are actively transported using carriers that utilise the stored energy in the proton electrochemical gradient. • • •

Pyruvate is taken up by a specific, low km transporter to bring it into the mitochondrial matrix for oxidation by the pyruvate dehydrogenase complex. The phosphate translocase is a symporter and the driving force for moving phosphate ions into the mitochondria is the proton motive force. The adenine nucleotide carrier is an antiporter and exchanges ADP and ATP across the inner membrane. The driving force is due to the ATP (-4) having a more negative charge than the ADP (-3) and thus it dissipates some of the electrical component of the proton electrochemical gradient.

The outcome of these transport processes using the proton electrochemical gradient is that more than 3 H+ are needed to make 1 ATP. Obviously this reduces the theoretical efficiency of the whole process and the likely maximum is closer to 28-30 ATP molecules. In practice the efficiency may be even lower due to the inner membrane of the mitochondria being slightly leaky to protons. Other factors may also dissipate the proton gradient creating an apparently leaky mitochondria. An uncoupling protein known as thermogenin is expressed in some cell types and is a channel that can transport protons. When this protein is active in the inner membrane it short circuits the coupling between the electron transport chain and ATP synthesis. The potential energy from the proton gradient is not used to make ATP but generates heat. This is particularly important in brown fat thermogenesis of newborn and hibernating mammals.

Fermentation Without oxygen, pyruvate (pyruvic acid) is not metabolized by cellular respiration but undergoes a process of fermentation. The pyruvate is not transported into the mitochondrion, but remains in the cytoplasm, where it is converted to waste products that may be removed from the cell. This serves the purpose of oxidizing the electron carriers so that they can perform glycolysis again and removing the excess pyruvate. This waste product varies depending on the organism. In skeletal muscles, the waste product is lactic acid. This type of fermentation is called lactic acid fermentation. In yeast, the waste products are ethanol and carbon dioxide. This type of fermentation is known as alcoholic or ethanol fermentation. The ATP generated in this process is made by substrate-level phosphorylation, which does not require oxygen. Fermentation is less efficient at using the energy from glucose since 2 ATP are produced per glucose, compared to the 38 ATP per glucose produced by aerobic respiration. This is because the waste products of fermentation still contain plenty of energy. Ethanol, for example, can be used in gasoline (petrol) solutions. Glycolytic ATP, however, is created more quickly. For prokaryotes to continue a rapid growth rate when they are shifted from an aerobic environment to an anaerobic environment, they must increase the rate of the glycolytic reactions. For multicellular organisms, during short bursts of strenuous activity, muscle cells use fermentation to supplement the ATP production from the slower aerobic respiration, so fermentation may be used by a cell even before the oxygen levels are depleted, as is the case in sports that do not require athletes to pace themselves, such as sprinting.

Anaerobic Respiration Anaerobic respiration is used by some microorganisms in which neither oxygen (aerobic respiration) nor pyruvate or pyruvate derivative (fermentation) is the final electron acceptor. Rather, an inorganic acceptor (for example, Sulfur) is used. Several distinct but linked metabolic pathways are used by cells to transfer the energy released by breakdown of fuel molecules into ATP and other small molecules used for energy (e.g. GTP, NADPH, FADH). Other pathways occurring in (most or) all living organisms include (but are not limited to): • • • • • • •

Fatty acid oxidation (β-oxidation) Gluconeogenesis Amino acid metabolism Urea cycle / Nitrogen metabolism Nucleotide metabolism Glycogen synthesis / Glycogen storage Pentose phosphate pathway (hexose monophosphate shunt)

• • •

Porphyrin synthesis (or heme synthesis) pathway Lipogenesis HMG-CoA reductase pathway

Synthesis of energetic compounds from non-living matter: • •

Photosynthesis (plants, algae, cyanobacteria) Chemosynthesis (some bacteria)

Chapter- 4

Glycolysis

Glycolysis overview Glycolysis (from glycose, an older term for glucose + -lysis degradation) is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+. The free energy released in this process is used to form the high-energy compounds ATP (adenosine triphosphate) and NADH (reduced nicotinamide adenine dinucleotide). Glycolysis is a definite sequence of ten reactions involving ten intermediate compounds (one of the steps involves two intermediates). The intermediates provide entry points to glycolysis. For example, most monosaccharides, such as fructose, glucose, and galactose, can be converted to one of these intermediates. The intermediates may also be directly useful. For example, the intermediate dihydroxyacetone phosphate (DHAP) is a source of the glycerol that combines with fatty acids to form fat. Glycolysis is thought to be the archetype of a universal metabolic pathway. It occurs, with variations, in nearly all organisms, both aerobic and anaerobic. The wide occurrence of glycolysis indicates that it is one of the most ancient known metabolic pathways.

The most common type of glycolysis is the Embden-Meyerhof-Parnas pathway (EMP pathway), which was first discovered by Gustav Embden, Otto Meyerhof and Jakub Karol Parnas. Glycolysis also refers to other pathways, such as the Entner–Doudoroff pathway and various heterofermentative and homofermentative pathways. However, the discussion here will be limited to the Embden-Meyerhof pathway.

Overview The overall reaction of glycolysis is: D-[Glucose]

[Pyruvate]

+ 2 [NAD]+ + 2 [ADP] + 2 [P]i

2

+ 2 [NADH] + 2 H+ + 2 [ATP]

The use of symbols in this equation makes it appear unbalanced with respect to oxygen atoms, hydrogen atoms, and charges. Atom balance is maintained by the two phosphate (Pi) groups: • •

each exists in the form of a hydrogen phosphate anion (HPO42-), dissociating to contribute 2 H+ overall each liberates an oxygen atom when it binds to an ADP (adenosine diphosphate) molecule, contributing 2 O overall

Charges are balanced by the difference between ADP and ATP. In the cellular environment, all three hydroxy groups of ADP dissociate into -O- and H+, giving ADP3-, and this ion tends to exist in an ionic bond with Mg2+, giving ADPMg-. ATP behaves identically except that it has four hydroxy groups, giving ATPMg2-. When these differences along with the true charges on the two phosphate groups are considered together, the net charges of -4 on each side are balanced.

Glycolysis For simple anaerobic fermentations, the metabolism of one molecule of glucose to two molecules of pyruvate has a net yield of two molecules of ATP. Most cells will then carry out further reactions to 'repay' the used NAD+ and produce a final product of ethanol or lactic acid. Many bacteria use inorganic compounds as hydrogen acceptors to regenerate the NAD+. Cells performing aerobic respiration synthesize much more ATP, but not as part of glycolysis. These further aerobic reactions use pyruvate and NADH + H+ from glycolysis. Eukaryotic aerobic respiration produces approximately 34 additional molecules of ATP for each glucose molecule, however most of these are produced by a vastly different mechanism to the substrate-level phosphorylation in glycolysis. The lower-energy production, per glucose, of anaerobic respiration relative to aerobic respiration, results in greater flux through the pathway under hypoxic (low-oxygen) conditions, unless alternative sources of anaerobically-oxidizable substrates, such as fatty acids, are found.

Elucidation of the pathway In 1860, Louis Pasteur discovered that microorganisms are responsible for fermentation. In 1897, Eduard Buchner found that extracts of certain cells can cause fermentation. In 1905, Arthur Harden and William Young determined that a heat-sensitive highmolecular-weight subcellular fraction (the enzymes) and a heat-insensitive lowmolecular-weight cytoplasm fraction (ADP, ATP and NAD+ and other cofactors) are required together for fermentation to proceed. The details of the pathway were eventually determined by 1940, with a major input from Otto Meyerhof and some years later by Luis Leloir. The biggest difficulties in determining the intricacies of the pathway were due to

the very short lifetime and low steady-state concentrations of the intermediates of the fast glycolytic reactions.

Sequence of reactions Preparatory phase The first five steps are regarded as the preparatory (or investment) phase, since they consume energy to convert the glucose into two three-carbon sugar phosphates (G3P). The first step in glycolysis is phosphorylation of glucose by a family of enzymes called hexokinases to form glucose 6-phosphate (G6P). This reaction consumes ATP, but it acts to keep the glucose concentration low, promoting continuous transport of glucose into the cell through the plasma membrane transporters. In addition, it blocks the glucose from leaking out the cell lacks transporters for G6P. Glucose may alternatively be from the phosphorolysis or hydrolysis of intracellular starch or glycogen. In animals, an isozyme of hexokinase called glucokinase is also used in the liver, which has a much lower affinity for glucose (Km in the vicinity of normal glycemia), and differs in regulatory properties. The different substrate affinity and alternate regulation of this enzyme are a reflection of the role of the liver in maintaining blood sugar levels. Cofactors: Mg2+

D-Glucose (Glc)

Hexokinase (HK) a transferase

ATP

H+ + ADP

α-D-Glucose-6phosphate (G6P)

G6P is then rearranged into fructose 6phosphate (F6P) by glucose phosphate isomerase. Fructose can also enter the glycolytic pathway by phosphorylation at this point. The change in structure is an isomerization, in which the G6P has been converted to F6P. The reaction requires an enzyme, phosphohexose isomerase, to proceed. This reaction is freely reversible under normal cell conditions. However, it is often driven forward because of a low concentration of F6P, which is constantly consumed during the next step of glycolysis. Under conditions of high F6P concentration, this reaction readily runs in reverse. This phenomenon can be explained through Le Chatelier's Principle.

α-D-Glucose 6-phosphate (G6P)

Phosphoglucose isomerase an isomerase

β-D-Fructose 6-phosphate (F6P)

The energy expenditure of another ATP in this step is justified in 2 ways: The glycolytic process (up to this step) is now irreversible, and the energy supplied destabilizes the molecule. Because the reaction catalyzed by Phosphofructokinase 1 (PFK-1) is coupled to the hydrolysis of ATP, an energetically favorable step, it is, in essence, irreversible, and a different pathway must be used to do the reverse conversion during gluconeogenesis. This makes the reaction a key regulatory point (see below). This is also the rate-limiting step. The same reaction can also be catalysed by pyrophosphatedependent phosphofructokinase (PFP or PPi-PFK), which is found in most plants, some bacteria, archea, and protists, but not in animals. This enzyme uses pyrophosphate (PPi) as a phosphate donor instead of ATP. It is a reversible reaction, increasing the flexibility of glycolytic metabolism. A rarer

β-D-Fructose 6-phosphate (F6P)

phosphofructokinase (PFK-1) a transferase ATP

H+ + ADP

β-D-Fructose 1,6bisphosphate (F1,6BP)

ADP-dependent PFK enzyme variant has been identified in archaean species. Cofactors: Mg2+

Destabilizing the molecule in the previous reaction allows the hexose ring to be split by aldolase into two triose sugars, dihydroxyacetone phosphate, a ketone, and glyceraldehyde 3-phosphate, an aldehyde. There are two classes of aldolases: class I aldolases, present in animals and plants, and class II aldolases, present in fungi and bacteria; the two classes use different mechanisms in cleaving the ketose ring.

fructose Dβ-D-Fructose 1,6bisphosphate glyceraldehyde bisphosphate (F1,6BP) aldolase (ALDO) 3-phosphate a lyase (GADP)

dihydroxyacetone phosphate (DHAP)

+

triosephosphate Dihydroxyacetone isomerase (TPI) phosphate (DHAP) an isomerase

D-glyceraldehyde 3phosphate (GADP)

Triosephosphate isomerase rapidly interconverts dihydroxyacetone phosphate with glyceraldehyde 3-phosphate (GADP) that proceeds further into glycolysis. This is advantageous, as it directs dihydroxyacetone phosphate down the same pathway as glyceraldehyde 3-phosphate, simplifying regulation.

Pay-off phase The second half of glycolysis is known as the pay-off phase, characterised by a net gain of the energy-rich molecules ATP and NADH. Since glucose leads to two triose sugars in the preparatory phase, each reaction in the pay-off phase occurs twice per glucose molecule. This yields 2 NADH molecules and 4 ATP molecules, leading to a net gain of 2 NADH molecules and 2 ATP molecules from the glycolytic pathway per glucose.

The triose sugars are dehydrogenated and inorganic phosphate is added to them, forming 1,3-bisphosphoglycerate. The hydrogen is used to reduce two molecules of NAD+, a hydrogen carrier, to give NADH + H+ for each triose. Hydrogen atom balance and charge balance are both maintained because the phosphate (Pi) group actually exists in the form of a hydrogen phosphate anion (HPO42), which dissociates to contribute the extra H+ ion and gives a net charge of -3 on both sides.

glyceraldehyde phosphate D-1,3glyceraldehyde 3- dehydrogenase bisphosphoglycerate phosphate (GADP) (GAPDH) (1,3BPG) an oxidoreductase NAD+ NADH + Pi + H+

This step is the enzymatic transfer of a phosphate group from 1,3bisphosphoglycerate to ADP by phosphoglycerate kinase, forming ATP and 3-phosphoglycerate. At this step, glycolysis has reached the break-even point: 2 molecules of ATP were consumed, and 2 new molecules have now been synthesized. This step, one of the two substrate-level phosphorylation steps, requires ADP; thus, when the cell has plenty of ATP (and little ADP), this reaction does not occur. Because ATP decays relatively quickly when it is not metabolized, this is an important regulatory point in the glycolytic pathway.

1,3phosphoglycerate 3bisphosphoglycerate kinase (PGK) phosphoglycerate (1,3-BPG) a transferase (3-P-G)

ADP actually exists as ADPMg-, and ATP as ATPMg2-, balancing the charges at -5 both sides. 2+

Cofactors: Mg

3-phosphoglycerate (3PG)

Phosphoglycerate mutase now forms 2phosphoglycerate.

ADP

ATP

phosphoglycerate kinase (PGK)

phosphoglycerate mutase (PGM) a mutase

2-phosphoglycerate (2PG)

Enolase next forms phosphoenolpyruvate from 2-phosphoglycerate.

2-phosphoglycerate (2PG)

enolase (ENO) a lyase

phosphoenolpyruvate (PEP)

H2O Cofactors: 2 Mg2+: one "conformational" ion to coordinate with the carboxylate group of the substrate, and one "catalytic" ion that participates in the dehydration.

enolase (ENO)

phosphoenolpyruvate (PEP) A final substrate-level phosphorylation now forms a molecule of pyruvate and a molecule of ATP by means of the enzyme pyruvate kinase. This serves as an additional regulatory step, similar to the phosphoglycerate kinase step.

pyruvate kinase (PK) a transferase

pyruvate (Pyr)

ADP + H+ ATP

Cofactors: Mg2+

Regulation Glycolysis is regulated by slowing down or speeding up certain steps in the glycolysis pathway. This is accomplished by inhibiting or activating the enzymes that are involved. The steps that are regulated may be determined by calculating the change in free energy, ΔG, for each step. If a step's products and reactants are in equilibrium, then the step is assumed to not be regulated. Since the change in free energy is zero for a system at equilibrium, any step with a free energy change near zero is not being regulated. If a step is being regulated, then that step's enzyme is not converting reactants into products as fast

as it could, resulting in a build-up of reactants, which would be converted to products if the enzyme were operating faster. Since the reaction is thermodynamically favorable, the change in free energy for the step will be negative. A step with a large negative change in free energy is assumed to be regulated.

Free energy changes Concentrations of metabolites in erythrocytes Compound Concentration / mM glucose 5.0 glucose-6-phosphate 0.083 fructose-6-phosphate 0.014 fructose-1,6-bisphosphate 0.031 dihydroxyacetone phosphate 0.14 glyceraldehyde-3-phosphate 0.019 1,3-bisphosphoglycerate 0.001 2,3-bisphosphoglycerate 4.0 3-phosphoglycerate 0.12 2-phosphoglycerate 0.03 phosphoenolpyruvate 0.023 pyruvate 0.051 ATP 1.85 ADP 0.14 Pi 1.0

The change in free energy for each step of glycolysis estimated from the concentration of metabolites in a erythrocyte. The change in free energy, ΔG, for each step in the glycolysis pathway can be calculated using ΔG = ΔG°' + RTln Q, where Q is the reaction quotient. This requires knowing the concentrations of the metabolites. All of these values are available for erythrocytes, with the exception of the concentrations of NAD+ and NADH. The ratio of NAD+ to NADH is approximately 1, which results in these concentrations canceling out in the reaction quotient. (Since NAD+ and NADH occur on opposite sides of the reactions, one will be in the numerator and the other in the denominator.) Using the measured concentrations of each step, and the standard free energy changes, the actual free energy change can be calculated. Change in free energy for each step of glycolysis ΔG°' / Step Reaction (kJ/mol) 423+ 1 glucose + ATP → glucose-6-phosphate + ADP + H -16.7 222 glucose-6-phosphate → fructose-6-phosphate 1.67 24fructose-6-phosphate + ATP → fructose-1,63 -14.2 bisphosphate4- + ADP3- + H+ fructose-1,6-bisphosphate4- → dihydroxyacetone 4 23.9 phosphate2- + glyceraldehyde-3-phosphate2dihydroxyacetone phosphate2- → glyceraldehyde-35 7.56 phosphate2glyceraldehyde-3-phosphate2- + Pi2- + NAD+ → 1,36 6.30 bisphosphoglycerate4- + NADH + H+ 1,3-bisphosphoglycerate4- + ADP3- → 3-18.9 7 phosphoglycerate3- + ATP48 3-phosphoglycerate3- → 2-phosphoglycerate34.4 339 2-phosphoglycerate → phosphoenolpyruvate + H2O 1.8 33+ phosphoenolpyruvate + ADP + H → pyruvate + 10 -31.7 ATP4-

ΔG / (kJ/mol) -34 -2.9 -19 -0.23 2.4 -1.29 0.09 0.83 1.1 -23.0

From measuring the physiological concentrations of metabolites in a erythrocyte it seems that about seven of the steps in glycolysis are in equilibrium for that cell type. Three of the steps — the ones with large negative free energy changes — are not in equilibrium and are referred to as irreversible; such steps are often subject to regulation. Step 5 in the figure is shown behind the other steps, because that step is a side-reaction that can decrease or increase the concentration of the intermediate glyceraldehyde-3phosphate. That compound is converted to dihydroxyacetone phosphate by the enzyme triose phosphate isomerase, which is a catalytically perfect enzyme; its rate is so fast that

the reaction can be assumed to be in equilibrium. The fact that ΔG is not zero indicates that the actual concentrations in the erythrocyte are not accurately known.

Biochemical logic The existence of more than one point of regulation indicates that intermediates between those points enter and leave the glycolysis pathway by other processes. For example, in the first regulated step, hexokinase converts glucose into glucose-6-phosphate. Instead of continuing through the glycolysis pathway, this intermediate can be converted into glucose storage molecules, such as glycogen or starch. The reverse reaction, breaking down, e.g., glycogen, produces mainly glucose-6-phosphate; very little free glucose is formed in the reaction. The glucose-6-phosphate so produced can enter glycolysis after the first control point. In the second regulated step (the third step of glycolysis), phosphofructokinase converts fructose-6-phosphate into fructose-1,6-bisphosphate, which then is converted into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. The dihydroxyacetone phosphate can be removed from glycolysis by conversion into glycerol-3-phosphate, which can be used to form triglycerides. On the converse, triglycerides can be broken down into fatty acids and glycerol; the latter, in turn, can be converted into dihydroxyacetone phosphate, which can enter glycolysis after the second control point.

Regulation The three regulated enzymes are hexokinase, phosphofructokinase, and pyruvate kinase. The flux through the glycolytic pathway is adjusted in response to conditions both inside and outside the cell. The rate in liver is regulated to meet major cellular needs: (1) the production of ATP, (2) the provision of building blocks for biosynthetic reactions, and (3) to lower blood glucose, one of the major functions of the liver. When blood sugar falls, glycolysis is halted in the liver to allow the reverse process, gluconeogenesis. In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are effectively irreversible in most organisms. In metabolic pathways, such enzymes are potential sites of control, and all three enzymes serve this purpose in glycolysis.

Hexokinase

Yeast hexokinase B. PDB 1IG8 In animals, regulation of blood glucose levels by the liver is a vital part of homeostasis. In liver cells, extra G6P (glucose-6-phosphate) may be converted to G1P for conversion to glycogen, or it is alternatively converted by glycolysis to acetyl-CoA and then citrate. Excess citrate is exported to the cytosol, where ATP citrate lyase will regenerate acetylCoA and OAA. The acetyl-CoA is then used for fatty acid synthesis and cholesterol synthesis, two important ways of utilizing excess glucose when its concentration is high in blood. Liver contains both hexokinase and glucokinase; the latter catalyses the phosphorylation of glucose to G6P and is not inhibited by G6P. Thus, it allows glucose to be converted into glycogen, fatty acids, and cholesterol even when hexokinase activity is low. This is important when blood glucose levels are high. During hypoglycemia, the glycogen can be converted back to G6P and then converted to glucose by the liverspecific enzyme glucose 6-phosphatase. This reverse reaction is an important role of liver cells to maintain blood sugars levels during fasting. This is critical for brain function, since the brain utilizes glucose as an energy source under most conditions.

Phosphofructokinase

Bacillus stearothermophilus phosphofructokinase. PDB 6PFK Phosphofructokinase is an important control point in the glycolytic pathway, since it is one of the irreversible steps and has key allosteric effectors, AMP and fructose 2,6bisphosphate (F2,6BP). Fructose 2,6-bisphosphate (F2,6BP) is a very potent activator of phosphofructokinase (PFK-1), which is synthesised when F6P is phosphorylated by a second phosphofructokinase (PFK2). In liver, when blood sugar is low and glucagon elevates cAMP, PFK2 is phosphorylated by protein kinase A. The phosphorylation inactivates PFK2, and another domain on this protein becomes active as fructose 2,6-bisphosphatase, which converts F2,6BP back to F6P. Both glucagon and epinephrine cause high levels of cAMP in the liver. The result of lower levels of liver fructose-2,6-bisphosphate is a decrease in activity of phosphofructokinase and an increase in activity of fructose 1,6bisphosphatase, so that gluconeogenesis (in essence, "glycolysis in reverse") is favored. This is consistent with the role of the liver in such situations, since the response of the liver to these hormones is to release glucose to the blood.

ATP competes with AMP for the allosteric effector site on the PFK enzyme. ATP concentrations in cells are much higher than those of AMP, typically 100-fold higher, but the concentration of ATP does not change more than about 10% under physiological conditions, whereas a 10% drop in ATP results in a 6-fold increase in AMP. Thus, the relevance of ATP as an allosteric effector is questionable. An increase in AMP is a consequence of a decrease in energy charge in the cell. Citrate inhibits phosphofructokinase when tested in vitro by enhancing the inhibitory effect of ATP. However, it is doubtful that this is a meaningful effect in vivo, because citrate in the cytosol is utilized mainly for conversion to acetyl-CoA for fatty acid and cholesterol synthesis.

Pyruvate kinase

Yeast pyruvate kinase. PDB 1A3W This enzyme catalyzes the last step of glycolysis, in which pyruvate and ATP are formed. Regulation of this enzyme is discussed in the main topic, pyruvate kinase.

Post-glycolysis processes The overall process of glycolysis is: glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O If glycolysis were to continue indefinitely, all of the NAD+ would be used up, and glycolysis would stop. To allow glycolysis to continue, organisms must be able to oxidize NADH back to NAD+.

Fermentation One method of doing this is to simply have the pyruvate do the oxidation; in this process, the pyruvate is converted to lactate (the conjugate base of lactic acid) in a process called lactic acid fermentation: pyruvate + NADH + H+ → lactate + NAD+ This process occurs in the bacteria involved in making yogurt (the lactic acid causes the milk to curdle). This process also occurs in animals under hypoxic (or partiallyanaerobic) conditions, found, for example, in overworked muscles that are starved of oxygen, or in infarcted heart muscle cells. In many tissues, this is a cellular last resort for energy; most animal tissue cannot maintain anaerobic respiration for an extended length of time. Some organisms, such as yeast, convert NADH back to NAD+ in a process called ethanol fermentation. In this process, the pyruvate is converted first to acetaldehyde and carbon dioxide, then to ethanol. Lactic acid fermentation and ethanol fermentation can occur in the absence of oxygen. This anaerobic fermentation allows many single-cell organisms to use glycolysis as their only energy source.

Anaerobic respiration In the above two examples of fermentation, NADH is oxidized by transferring two electrons to pyruvate. However, anaerobic bacteria use a wide variety of compounds as the terminal electron acceptors in cellular respiration: nitrogenous compounds, such as nitrates and nitrites; sulfur compounds, such as sulfates, sulfites, sulfur dioxide, and elemental sulfur; carbon dioxide; iron compounds; manganese compounds; cobalt compounds; and uranium compounds.

Aerobic respiration In aerobic organisms, a complex mechanism has been created to use the oxygen in air as the final electron acceptor of respiration. • • • •

First, pyruvate is converted to acetyl-CoA and CO2 within the mitochondria in a process called pyruvate decarboxylation. Second, the acetyl-CoA enters the citric acid cycle, or Krebs Cycle, where it is fully oxidized to carbon dioxide and water, producing yet more NADH. Third, the NADH is oxidized to NAD+ by the electron transport chain, using oxygen as the final electron acceptor. This process creates a "hydrogen ion gradient" across the inner membrane of the mitochondria. Fourth, the proton gradient is used to produce a large amount of ATP in a process called oxidative phosphorylation.

Intermediates for other pathways Here we concentrates on the catabolic role of glycolysis with regard to converting potential chemical energy to usable chemical energy during the oxidation of glucose to pyruvate. However, many of the metabolites in the glycolytic pathway are also used by anabolic pathways, and, as a consequence, flux through the pathway is critical to maintain a supply of carbon skeletons for biosynthesis. In addition, not all carbon entering the pathway leaves as pyruvate and may be extracted at earlier stages to provide carbon compounds for other pathways. These metabolic pathways are all strongly reliant on glycolysis as a source of metabolites: • • • •

Gluconeogenesis Lipid metabolism Pentose phosphate pathway Citric acid cycle, which in turn leads to: • • •

Amino acid synthesis Nucleotide synthesis Tetrapyrrole synthesis

From an anabolic metabolism perspective, the NADH has a role to drive synthetic reactions, doing so by directly or indirectly reducing the pool of NADP+ in the cell to NADPH, which is another important reducing agent for biosynthetic pathways in a cell.

Glycolysis in disease Genetic diseases Glycolytic mutations are generally rare due to importance of the metabolic pathway, this means that the majority of occurring mutations result in an inability for the cell to respire, and therefore cause the death of the cell at an early stage. However, some mutations are seen with one notable example being Pyruvate kinase deficiency, leading to chronic hemolytic anemia.

Cancer Malignant rapidly-growing tumor cells typically have glycolytic rates that are up to 200 times higher than those of their normal tissues of origin. This phenomenon was first described in 1930 by Otto Warburg and is referred to as the Warburg effect. The Warburg hypothesis claims that cancer is primarily caused by dysfunctionality in mitochondrial metabolism, rather than because of uncontrolled growth of cells. A number of theories have been advanced to explain the Warburg effect. This high glycolysis rate has important medical applications, as high aerobic glycolysis by malignant tumors is utilized clinically to diagnose and monitor treatment responses of cancers by imaging uptake of 2-18F-2-deoxyglucose (FDG) (a radioactive modified hexokinase substrate) with positron emission tomography (PET). There is ongoing research to affect mitochondrial metabolism and treat cancer by reducing glycolysis and thus starving cancerous cells in various new ways, including a ketogenic diet.

Alzheimer's disease Disfunctioning glycolysis or glucose metabolism in fronto-temporo-parietal and cingulate cortices has been associated with Alzheimer's disease, probably due to the decreased amyloid β (1-42) (Aβ42) and increased tau, phosphorylated tau in cerebrospinal fluid (CSF)

Alternative nomenclature Some of the metabolites in glycolysis have alternative names and nomenclature. In part, this is because some of them are common to other pathways, such as the Calvin cycle.

Given above 1 glucose Glc 3 fructose 6-phosphate F6P 4 fructose 1,6-bisphosphate F1,6BP dihydroxyacetone 5 DHAP phosphate glyceraldehyde 36 GADP phosphate

Alternative names

Alternative nomenclature

dextrose fructose 1,6-diphosphate FBP, FDP, F1,6DP glycerone phosphate 3-phosphoglyceraldehyde

glycerate 1,3bisphosphate, 7 1,3-bisphosphoglycerate 1,3BPG glycerate 1,3diphosphate, 1,3-diphosphoglycerate 8 3-phosphoglycerate glycerate 3-phosphate 3PG 9 2-phosphoglycerate glycerate 2-phosphate 2PG 10 phosphoenolpyruvate PEP 11 pyruvate pyruvic acid Pyr

PGAL, G3P, GALP,GAP,TP

PGAP, BPG, DPG

PGA, GP

Chapter- 5

Citric Acid Cycle

Overview of the citric acid cycle The citric acid cycle — also known as the tricarboxylic acid cycle (TCA cycle), the Krebs cycle, or the Szent-Györgyi-Krebs cycle — is a series of enzyme-catalysed chemical reactions, which is of central importance in all living cells that use oxygen as part of cellular respiration. In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion. The components and reactions of the citric acid cycle were established by seminal work from Albert Szent-Györgyi and Hans Krebs.

In aerobic organisms, the citric acid cycle is part of a metabolic pathway involved in the chemical conversion of carbohydrates, fats and proteins into carbon dioxide and water to generate a form of usable energy. Other relevant reactions in the pathway include those in glycolysis and pyruvate oxidation before the citric acid cycle, and oxidative phosphorylation after it. In addition, it provides precursors for many compounds including some amino acids and is therefore functional even in cells performing fermentation.

A simplified view of the process • •

• • •

The citric acid cycle begins with the transfer of a two-carbon acetyl group from acetyl-CoA to the four-carbon acceptor compound (oxaloacetate) to form a sixcarbon compound (citrate). The citrate then goes through a series of chemical transformations, losing two carboxyl groups as CO2. The carbons lost as CO2 originate from what was oxaloacetate, not directly from acetyl-CoA. The carbons donated by acetyl-CoA become part of the oxaloacetate carbon backbone after the first turn of the citric acid cycle. Loss of the acetyl-CoA-donated carbons as CO2 requires several turns of the citric acid cycle. However, because of the role of the citric acid cycle in anabolism, they may not be lost, since many TCA cycle intermediates are also used as precursors for the biosynthesis of other molecules. 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. Electrons are also transferred to the electron acceptor Q, forming QH2. At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues.

Steps Two carbon atoms are oxidized to CO2, the energy from these reactions being transferred to other metabolic processes by GTP (or ATP), and as electrons in NADH and QH2. The NADH generated in the TCA cycle may later donate its electrons in oxidative phosphorylation to drive ATP synthesis; FADH2 is covalently attached to succinate dehydrogenase, an enzyme functioning both in the TCA cycle and the mitochondrial electron transport chain in oxidative phosphorylation. FADH2, therefore, facilitates transfer of electrons to coenzyme Q, which is the final electron acceptor of the reaction catalyzed by the Succinate:ubiquinone oxidoreductase complex, also acting as an intermediate in the electron transport chain. The citric acid cycle is continuously supplied with new carbon in the form of acetyl-CoA, entering at step 1 below.

Substrates

Products

Oxaloacetate + Citrate + 1 Acetyl CoA + CoA-SH H2O 2 Citrate

cis-Aconitate + H2O

Enzyme Citrate synthase

Reaction type Aldol condensation

Dehydration Aconitase

cis-Aconitate + Isocitrate Hydration H2O Oxalosuccinate Isocitrate + Oxidation 4 + NAD+ NADH + H + Isocitrate αdehydrogenase Ketoglutarate 5 Oxalosuccinate Decarboxylation + CO2 3

Comment rate-limiting stage (irreversible), extends the 4C oxaloacetate to a 6C molecule reversible isomerisation generates NADH (equivalent of 2.5 ATP) irreversible stage, generates a 5C molecule

irreversible stage, generates NADH Succinyl-CoA (equivalent of 2.5 α-Ketoglutarate Oxidative + ATP), NADH + H+ + dehydrogenase decarboxylation regenerates the 4C CO2 chain (CoA excluded) or ADP→ATP Succinyl-CoA Succinate + instead of Succinyl-CoA substrate-level CoA-SH + 7 + GDP→GTP, synthetase phosphorylation GTP GDP + Pi generates 1 ATP or equivalent uses FAD as a prosthetic group (FAD→FADH2 in Fumarate + the first step of the Succinate + Succinate 8 ubiquinol Oxidation reaction) in the ubiquinone (Q) dehydrogenase (QH2) enzyme, generates the equivalent of 1.5 ATP Fumarate + H2O addition 9 L-Malate Fumarase H2O (hydration) generates NADH L-Malate + Oxaloacetate + Malate 10 Oxidation (equivalent of 2.5 NAD+ NADH + H+ dehydrogenase ATP) αKetoglutarate 6 + NAD+ + CoA-SH

Mitochondria in animals, including humans, possess two succinyl-CoA synthetases: one that produces GTP from GDP, and another that produces ATP from ADP. Plants have the type that produces ATP (ADP-forming succinyl-CoA synthetase). Several of the enzymes in the cycle may be loosely-associated in a multienzyme protein complex within the mitochondrial matrix. The GTP that is formed by GDP-forming succinyl-CoA synthetase may be utilized by nucleoside-diphosphate kinase to form ATP (the catalyzed reaction is GTP + ADP → GDP + ATP).

Products Products of the first turn of the cycle are: one GTP (or ATP), three NADH, one QH2, two CO2. Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule. Therefore, at the end of two cycles, the products are: two GTP, six NADH, two QH2, and four CO2 Description

Reactants

Products → CoA-SH + 3 Acetyl-CoA + 3 The sum of all reactions in the citric acid cycle NADH + 3 H+ + + NAD + Q + GDP + is: QH2 + GTP + 2 Pi + 2 H2O CO2 Combining the reactions occurring during the → 4 NADH + 3 H+ Pyruvate ion + 4 pyruvate oxidation with those occurring during NAD+ + Q + GDP + + QH2 + GTP + 3 the citric acid cycle, the following overall Pi + 2 H2O CO2 pyruvate oxidation reaction is obtained: Combining the above reaction with the ones → 10 NADH + 10 occurring in the course of glycolysis, the Glucose + 10 NAD+ + H + 2 QH2 + 2 following overall glucose oxidation reaction + 2 Q + 2 ADP + 2 ATP + 2 GTP + 6 (excluding reactions in the respiratory chain) is GDP + 4 Pi + 2 H2O CO2 obtained: The above reactions are balanced if Pi represents the H2PO4- ion, ADP and GDP the ADP2- and GDP2- ions, respectively, and ATP and GTP the ATP3- and GTP3- ions, respectively. The total number of ATP obtained after complete oxidation of one glucose in glycolysis, citric acid cycle, and oxidative phosphorylation is estimated to be between 30 and 38. A recent assessment of the total ATP yield with the updated proton-to-ATP ratios provides an estimate of 29.85 ATP per glucose molecule.

Regulation The regulation of the TCA cycle is largely determined by substrate availability and product inhibition. NADH, a product of all dehydrogenases in the TCA cycle with the exception of succinate dehydrogenase, inhibits pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and also citrate synthase. Acetyl-coA inhibits pyruvate dehydrogenase, while succinyl-CoA inhibits succinyl-CoA synthetase and citrate synthase. When tested in vitro with TCA enzymes, ATP inhibits citrate synthase and α-ketoglutarate dehydrogenase; however, ATP levels do not change more than 10% in vivo between rest and vigorous exercise. There is no known allosteric mechanism that can account for large changes in reaction rate from an allosteric effector whose concentration changes less than 10%. Calcium is used as a regulator. It activates pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. This increases the reaction rate of many of the steps in the cycle, and therefore increases flux throughout the pathway. Citrate is used for feedback inhibition, as it inhibits phosphofructokinase, an enzyme involved in glycolysis that catalyses formation of fructose 1,6-bisphosphate,a precursor of pyruvate. This prevents a constant high rate of flux when there is an accumulation of citrate and a decrease in substrate for the enzyme. Recent work has demonstrated an important link between intermediates of the citric acid cycle and the regulation of hypoxia-inducible factors (HIF). HIF plays a role in the regulation of oxygen homeostasis, and is a transcription factor that targets angiogenesis, vascular remodeling, glucose utilization, iron transport and apoptosis. HIF is synthesized consititutively, and hydroxylation of at least one of two critical proline residues mediates their interaction with the von Hippel Lindau E3 ubiquitin ligase complex, which targets them for rapid degradation. This reaction is calalysed by prolyl 4-hydroxylases. Fumarate and succinate have been identified as potent inhibitors of prolyl hydroxylases, thus leading to the stabilisation of HIF.

Major metabolic pathways converging on the TCA cycle Several catabolic pathways converge on the TCA cycle. Reactions that form intermediates of the TCA cycle in order to replenish them (especially during the scarcity of the intermediates) are called anaplerotic reactions. The citric acid cycle is the third step in carbohydrate catabolism (the breakdown of sugars). Glycolysis breaks glucose (a six-carbon-molecule) down into pyruvate (a threecarbon molecule). In eukaryotes, pyruvate moves into the mitochondria. It is converted into acetyl-CoA by decarboxylation and enters the citric acid cycle.

In protein catabolism, proteins are broken down by proteases into their constituent amino acids. The carbon backbone of these amino acids can become a source of energy by being converted to acetyl-CoA and entering into the citric acid cycle. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In many tissues, especially heart tissue, fatty acids are broken down through a process known as beta oxidation, which results in acetyl-CoA, which can be used in the citric acid cycle. Beta oxidation of fatty acids with an odd number of methylene groups produces propionyl CoA, which is then converted into succinyl-CoA and fed into the citric acid cycle. The total energy gained from the complete breakdown of one molecule of glucose by glycolysis, the citric acid cycle, and oxidative phosphorylation equals about 30 ATP molecules, in eukaryotes. The citric acid cycle is called an amphibolic pathway because it participates in both catabolism and anabolism.

Interactive pathway map

Citric acid cycle

Chapter- 6

Oxidative Phosphorylation

The electron transport chain in the mitochondrion is the site of oxidative phosphorylation in eukaryotes. The NADH and succinate generated in the citric acid cycle are oxidized, releasing energy to power the ATP synthase. Oxidative phosphorylation is a metabolic pathway that uses energy released by the oxidation of nutrients to produce adenosine triphosphate (ATP). Although the many

forms of life on earth use a range of different nutrients, almost all carry out oxidative phosphorylation to produce ATP, the molecule that supplies energy to metabolism. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis. During oxidative phosphorylation, electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions. These redox reactions release energy, which is used to form ATP. In eukaryotes, these redox reactions are carried out by a series of protein complexes within mitochondria, whereas, in prokaryotes, these proteins are located in the cells' inner membranes. These linked sets of proteins are called electron transport chains. In eukaryotes, five main protein complexes are involved, whereas in prokaryotes many different enzymes are present, using a variety of electron donors and acceptors. The energy released by electrons flowing through this electron transport chain is used to transport protons across the inner mitochondrial membrane, in a process called chemiosmosis. This generates potential energy in the form of a pH gradient and an electrical potential across this membrane. This store of energy is tapped by allowing protons to flow back across the membrane and down this gradient, through a large enzyme called ATP synthase. This enzyme uses this energy to generate ATP from adenosine diphosphate (ADP), in a phosphorylation reaction. This reaction is driven by the proton flow, which forces the rotation of a part of the enzyme; the ATP synthase is a rotary mechanical motor. Although oxidative phosphorylation is a vital part of metabolism, it produces reactive oxygen species such as superoxide and hydrogen peroxide, which lead to propagation of free radicals, damaging cells and contributing to disease and, possibly, aging (senescence). The enzymes carrying out this metabolic pathway are also the target of many drugs and poisons that inhibit their activities.

Overview of energy transfer by chemiosmosis Oxidative phosphorylation works by using energy-releasing chemical reactions to drive energy-requiring reactions: The two sets of reactions are said to be coupled. This means one cannot occur without the other. The flow of electrons through the electron transport chain, from electron donors such as NADH to electron acceptors such as oxygen, is an exergonic process – it releases energy, whereas the synthesis of ATP is an endergonic process, which requires an input of energy. Both the electron transport chain and the ATP synthase are embedded in a membrane, and energy is transferred from electron transport chain to the ATP synthase by movements of protons across this membrane, in a process called chemiosmosis. In practice, this is like a simple electric circuit, with a current of protons being driven from the negative N-side of the membrane to the positive P-side by the proton-pumping enzymes of the electron transport chain. These enzymes are like a battery, as they perform work to drive current through the circuit. The movement of protons creates an electrochemical gradient across the membrane, which is often called the proton-motive force. This gradient has two components: a difference in proton

concentration (a H+ gradient) and a difference in electric potential, with the N-side having a negative charge. The energy is stored largely as the difference of electric potentials in mitochondria, but also as a pH gradient in chloroplasts. ATP synthase releases this stored energy by completing the circuit and allowing protons to flow down the electrochemical gradient, back to the N-side of the membrane. This enzyme is like an electric motor as it uses the proton-motive force to drive the rotation of part of its structure and couples this motion to the synthesis of ATP. The amount of energy released by oxidative phosphorylation is high, compared with the amount produced by anaerobic fermentation. Glycolysis produces only 2 ATP molecules, but somewhere between 30 and 36 ATPs are produced by the oxidative phosphorylation of the 10 NADH and 2 succinate molecules made by converting one molecule of glucose to carbon dioxide and water. This ATP yield is the theoretical maximum value; in practice, some protons leak across the membrane, lowering the yield of ATP.

Electron and proton transfer molecules

Reduction of coenzyme Q from its ubiquinone form (Q) to the reduced ubiquinol form (QH2). The electron transport chain carries both protons and electrons, passing electrons from donors to acceptors, and transporting protons across a membrane. These processes use both soluble and protein-bound transfer molecules. In mitochondria, electrons are transferred within the intermembrane space by the water-soluble electron transfer protein cytochrome c. This carries only electrons, and these are transferred by the reduction and oxidation of an iron atom that the protein holds within a heme group in its structure.

Cytochrome c is also found in some bacteria, where it is located within the periplasmic space. Within the inner mitochondrial membrane, the lipid-soluble electron carrier coenzyme Q10 (Q) carries both electrons and protons by a redox cycle. This small benzoquinone molecule is very hydrophobic, so it diffuses freely within the membrane. When Q accepts two electrons and two protons, it becomes reduced to the ubiquinol form (QH2); when QH2 releases two electrons and two protons, it becomes oxidized back to the ubiquinone (Q) form. As a result, if two enzymes are arranged so that Q is reduced on one side of the membrane and QH2 oxidized on the other, ubiquinone will couple these reactions and shuttle protons across the membrane. Some bacterial electron transport chains use different quinones, such as menaquinone, in addition to ubiquinone. Within proteins, electrons are transferred between flavin cofactors, iron–sulfur clusters, and cytochromes. There are several types of iron–sulfur cluster. The simplest kind found in the electron transfer chain consists of two iron atoms joined by two atoms of inorganic sulfur; these are called [2Fe–2S] clusters. The second kind, called [4Fe–4S], contains a cube of four iron atoms and four sulfur atoms. Each iron atom in these clusters is coordinated by an additional amino acid, usually by the sulfur atom of cysteine. Metal ion cofactors undergo redox reactions without binding or releasing protons, so in the electron transport chain they serve solely to transport electrons through proteins. Electrons move quite long distances through proteins by hopping along chains of these cofactors. This occurs by quantum tunnelling, which is rapid over distances of less than 1.4×10−9 m.

Eukaryotic electron transport chains Many catabolic biochemical processes, such as glycolysis, the citric acid cycle, and beta oxidation, produce the reduced coenzyme NADH. This coenzyme contains electrons that have a high transfer potential; in other words, they will release a large amount of energy upon oxidation. However, the cell does not release this energy all at once, as this would be an uncontrollable reaction. Instead, the electrons are removed from NADH and passed to oxygen through a series of enzymes that each release a small amount of the energy. This set of enzymes, consisting of complexes I through IV, is called the electron transport chain and is found in the inner membrane of the mitochondrion. Succinate is also oxidized by the electron transport chain, but feeds into the pathway at a different point. In eukaryotes, the enzymes in this electron transport system use the energy released from the oxidation of NADH to pump protons across the inner membrane of the mitochondrion. This causes protons to build up in the intermembrane space, and generates an electrochemical gradient across the membrane. The energy stored in this potential is then used by ATP synthase to produce ATP. Oxidative phosphorylation in the eukaryotic mitochondrion is the best-understood example of this process. The mitochondrion is present in almost all eukaryotes, with the exception of anaerobic protozoa such as Trichomonas vaginalis that instead reduce protons to hydrogen in a remnant mitochondrion called a hydrogenosome.

Typical respiratory enzymes and substrates in eukaryotes. Midpoint potential Respiratory enzyme Redox pair (Volts) + NADH dehydrogenase NAD / NADH −0.32 Succinate dehydrogenase FMN or FAD / FMNH2 or FADH2 −0.20 Cytochrome bc1 complex Coenzyme Q10ox / Coenzyme Q10red +0.06 Cytochrome bc1 complex Cytochrome box / Cytochrome bred +0.12 Complex IV Cytochrome cox / Cytochrome cred +0.22 Complex IV Cytochrome aox / Cytochrome ared +0.29 − Complex IV O2 / HO +0.82 Conditions: pH = 7

NADH-coenzyme Q oxidoreductase (complex I)

Complex I or NADH-Q oxidoreductase. The abbreviations are discussed in the text. In all diagrams of respiratory complexes here, the matrix is at the bottom, with the intermembrane space above.

NADH-coenzyme Q oxidoreductase, also known as NADH dehydrogenase or complex I, is the first protein in the electron transport chain. Complex I is a giant enzyme with the mammalian complex I having 46 subunits and a molecular mass of about 1,000 kilodaltons (kDa). The structure is known in detail only from a bacterium; in most organisms the complex resembles a boot with a large “ball” poking out from the membrane into the mitochondrion. The genes that encode the individual proteins are contained in both the cell nucleus and the mitochondrial genome, as is the case for many enzymes present in the mitochondrion. The reaction that is catalyzed by this enzyme is the two electron reduction by NADH of coenzyme Q10 or ubiquinone (represented as Q in the equation below), a lipid-soluble quinone that is found in the mitochondrion membrane:

The start of the reaction, and indeed of the entire electron chain, is the binding of a NADH molecule to complex I and the donation of two electrons. The electrons enter complex I via a prosthetic group attached to the complex, flavin mononucleotide (FMN). The addition of electrons to FMN converts it to its reduced form, FMNH2. The electrons are then transferred through a series of iron–sulfur clusters: the second kind of prosthetic group present in the complex. There are both [2Fe–2S] and [4Fe–4S] iron–sulfur clusters in complex I. As the electrons pass through this complex, four protons are pumped from the matrix into the intermembrane space. Exactly how this occurs is unclear, but it seems to involve conformational changes in complex I that cause the protein to bind protons on the N-side of the membrane and release them on the P-side of the membrane. Finally, the electrons are transferred from the chain of iron–sulfur clusters to a ubiquinone molecule in the membrane. Reduction of ubiquinone also contributes to the generation of a proton gradient, as two protons are taken up from the matrix as it is reduced to ubiquinol (QH2).

Succinate-Q oxidoreductase (complex II)

Complex II: Succinate-Q oxidoreductase Succinate-Q oxidoreductase, also known as complex II or succinate dehydrogenase, is a second entry point to the electron transport chain. It is unusual because it is the only enzyme that is part of both the citric acid cycle and the electron transport chain. Complex II consists of four protein subunits and contains a bound flavin adenine dinucleotide (FAD) cofactor, iron–sulfur clusters, and a heme group that does not participate in electron transfer to coenzyme Q, but is believed to be important in decreasing production of reactive oxygen species. It oxidizes succinate to fumarate and reduces ubiquinone. As this reaction releases less energy than the oxidation of NADH, complex II does not transport protons across the membrane and does not contribute to the proton gradient.

In some eukaryotes, such as the parasitic worm Ascaris suum, an enzyme similar to complex II, fumarate reductase (menaquinol:fumarate oxidoreductase, or QFR), operates in reverse to oxidize ubiquinol and reduce fumarate. This allows the worm to survive in the anaerobic environment of the large intestine, carrying out anaerobic oxidative phosphorylation with fumarate as the electron acceptor. Another unconventional function of complex II is seen in the malaria parasite Plasmodium falciparum. Here, the reversed action of complex II as an oxidase is important in regenerating ubiquinol, which the parasite uses in an unusual form of pyrimidine biosynthesis.

Electron transfer flavoprotein-Q oxidoreductase Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Q oxidoreductase), also known as electron transferring-flavoprotein dehydrogenase, is a third entry point to the electron transport chain. It is an enzyme that accepts electrons from electron-transferring flavoprotein in the mitochondrial matrix, and uses these electrons to reduce ubiquinone. This enzyme contains a flavin and a [4Fe–4S] cluster, but, unlike the other respiratory complexes, it attaches to the surface of the membrane and does not cross the lipid bilayer.

In mammals, this metabolic pathway is important in beta oxidation of fatty acids and catabolism of amino acids and choline, as it accepts electrons from multiple acetyl-CoA dehydrogenases. In plants, ETF-Q oxidoreductase is also important in the metabolic responses that allow survival in extended periods of darkness.

Q-cytochrome c oxidoreductase (complex III)

The two electron transfer steps in complex III: Q-cytochrome c oxidoreductase. After each step, Q (in the upper part of the figure) leaves the enzyme.

Q-cytochrome c oxidoreductase is also known as cytochrome c reductase, cytochrome bc1 complex, or simply complex III. In mammals, this enzyme is a dimer, with each subunit complex containing 11 protein subunits, an [2Fe-2S] iron–sulfur cluster and three cytochromes: one cytochrome c1 and two b cytochromes. A cytochrome is a kind of electron-transferring protein that contains at least one heme group. The iron atoms inside complex III’s heme groups alternate between a reduced ferrous (+2) and oxidized ferric (+3) state as the electrons are transferred through the protein. The reaction catalyzed by complex III is the oxidation of one molecule of ubiquinol and the reduction of two molecules of cytochrome c, a heme protein loosely associated with the mitochondrion. Unlike coenzyme Q, which carries two electrons, cytochrome c carries only one electron.

As only one of the electrons can be transferred from the QH2 donor to a cytochrome c acceptor at a time, the reaction mechanism of complex III is more elaborate than those of the other respiratory complexes, and occurs in two steps called the Q cycle. In the first step, the enzyme binds three substrates, first, QH2, which is then oxidized, with one electron being passed to the second substrate, cytochrome c. The two protons released from QH2 pass into the intermembrane space. The third substrate is Q, which accepts the second electron from the QH2 and is reduced to Q.-, which is the ubisemiquinone free radical. The first two substrates are released, but this ubisemiquinone intermediate remains bound. In the second step, a second molecule of QH2 is bound and again passes its first electron to a cytochrome c acceptor. The second electron is passed to the bound ubisemiquinone, reducing it to QH2 as it gains two protons from the mitochondrial matrix. This QH2 is then released from the enzyme. As coenzyme Q is reduced to ubiquinol on the inner side of the membrane and oxidized to ubiquinone on the other, a net transfer of protons across the membrane occurs, adding to the proton gradient. The rather complex two-step mechanism by which this occurs is important, as it increases the efficiency of proton transfer. If, instead of the Q cycle, one molecule of QH2 were used to directly reduce two molecules of cytochrome c, the efficiency would be halved, with only one proton transferred per cytochrome c reduced.

Cytochrome c oxidase (complex IV)

Complex IV: cytochrome c oxidase Cytochrome c oxidase, also known as complex IV, is the final protein complex in the electron transport chain. The mammalian enzyme has an extremely complicated structure and contains 13 subunits, two heme groups, as well as multiple metal ion cofactors – in all three atoms of copper, one of magnesium and one of zinc. This enzyme mediates the final reaction in the electron transport chain and transfers electrons to oxygen, while pumping protons across the membrane. The final electron acceptor oxygen, which is also called the terminal electron acceptor, is reduced to water in this step. Both the direct pumping of protons and the consumption of matrix protons in

the reduction of oxygen contribute to the proton gradient. The reaction catalyzed is the oxidation of cytochrome c and the reduction of oxygen:

Alternative reductases and oxidases Many eukaryotic organisms have electron transport chains that differ from the muchstudied mammalian enzymes described above. For example, plants have alternative NADH oxidases, which oxidize NADH in the cytosol rather than in the mitochondrial matrix, and pass these electrons to the ubiquinone pool. These enzymes do not transport protons, and, therefore, reduce ubiquinone without altering the electrochemical gradient across the inner membrane. Another example of a divergent electron transport chain is the alternative oxidase, which is found in plants, as well as some fungi, protists, and possibly some animals. This enzyme transfers electrons directly from ubiquinol to oxygen. The electron transport pathways produced by these alternative NADH and ubiquinone oxidases have lower ATP yields than the full pathway. The advantages produced by a shortened pathway are not entirely clear. However, the alternative oxidase is produced in response to stresses such as cold, reactive oxygen species, and infection by pathogens, as well as other factors that inhibit the full electron transport chain. Alternative pathways might, therefore, enhance an organisms' resistance to injury, by reducing oxidative stress.

Organization of complexes The original model for how the respiratory chain complexes are organized was that they diffuse freely and independently in the mitochondrial membrane. However, recent data suggest that the complexes might form higher-order structures called supercomplexes or "respirasomes." In this model, the various complexes exist as organized sets of interacting enzymes. These associations might allow channeling of substrates between the various enzyme complexes, increasing the rate and efficiency of electron transfer. Within such mammalian supercomplexes, some components would be present in higher amounts than others, with some data suggesting a ratio between complexes I/II/III/IV and the ATP synthase of approximately 1:1:3:7:4. However, the debate over this supercomplex hypothesis is not completely resolved, as some data do not appear to fit with this model.

Prokaryotic electron transport chains In contrast to the general similarity in structure and function of the electron transport chains in eukaryotes, bacteria and archaea possess a large variety of electron-transfer enzymes. These use an equally wide set of chemicals as substrates. In common with eukaryotes, prokaryotic electron transport uses the energy released from the oxidation of

a substrate to pump ions across a membrane and generate an electrochemical gradient. In the bacteria, oxidative phosphorylation in Escherichia coli is understood in most detail, while archaeal systems are at present poorly understood. The main difference between eukaryotic and prokaryotic oxidative phosphorylation is that bacteria and archaea use many different substances to donate or accept electrons. This allows prokaryotes to grow under a wide variety of environmental conditions. In E. coli, for example, oxidative phosphorylation can be driven by a large number of pairs of reducing agents and oxidizing agents, which are listed below. The midpoint potential of a chemical measures how much energy is released when it is oxidized or reduced, with reducing agents having negative potentials and oxidizing agents positive potentials. Respiratory enzymes and substrates in E. coli. Respiratory enzyme Formate dehydrogenase Hydrogenase NADH dehydrogenase Glycerol-3-phosphate dehydrogenase

Redox pair Bicarbonate / Formate Proton / Hydrogen NAD+ / NADH

(Volts) −0.43 −0.42 −0.32

DHAP / Gly-3-P

−0.19

Acetate + Carbon dioxide / Pyruvate Lactate dehydrogenase Pyruvate / Lactate 2-oxoacid + ammonia / D-amino D-amino acid dehydrogenase acid Glucose dehydrogenase Gluconate / Glucose Succinate dehydrogenase Fumarate / Succinate Ubiquinol oxidase Oxygen / Water Nitrate reductase Nitrate / Nitrite Nitrite reductase Nitrite / Ammonia Dimethyl sulfoxide reductase DMSO / DMS Trimethylamine N-oxide reductase TMAO / TMA Fumarate reductase Fumarate / Succinate Pyruvate oxidase

Midpoint potential

? −0.19 ? −0.14 +0.03 +0.82 +0.42 +0.36 +0.16 +0.13 +0.03

As shown above, E. coli can grow with reducing agents such as formate, hydrogen, or lactate as electron donors, and nitrate, DMSO, or oxygen as acceptors. The larger the difference in midpoint potential between an oxidizing and reducing agent, the more energy is released when they react. Out of these compounds, the succinate/fumarate pair is unusual, as its midpoint potential is close to zero. Succinate can therefore be oxidized to fumarate if a strong oxidizing agent such as oxygen is available, or fumarate can be

reduced to succinate using a strong reducing agent such as formate. These alternative reactions are catalyzed by succinate dehydrogenase and fumarate reductase, respectively. Some prokaryotes use redox pairs that have only a small difference in midpoint potential. For example, nitrifying bacteria such as Nitrobacter oxidize nitrite to nitrate, donating the electrons to oxygen. The small amount of energy released in this reaction is enough to pump protons and generate ATP, but not enough to produce NADH or NADPH directly for use in anabolism. This problem is solved by using a nitrite oxidoreductase to produce enough proton-motive force to run part of the electron transport chain in reverse, causing complex I to generate NADH. Prokaryotes control their use of these electron donors and acceptors by varying which enzymes are produced, in response to environmental conditions. This flexibility is possible because different oxidases and reductases use the same ubiquinone pool. This allows many combinations of enzymes to function together, linked by the common ubiquinol intermediate. These respiratory chains therefore have a modular design, with easily interchangeable sets of enzyme systems. In addition to this metabolic diversity, prokaryotes also possess a range of isozymes – different enzymes that catalyze the same reaction. For example, in E. coli, there are two different types of ubiquinol oxidase using oxygen as an electron acceptor. Under highly aerobic conditions, the cell uses an oxidase with a low affinity for oxygen that can transport two protons per electron. However, if levels of oxygen fall, they switch to an oxidase that transfers only one proton per electron, but has a high affinity for oxygen.

ATP synthase (complex V)

ATP synthase. The FO proton channel and stalk are shown in blue, the F1 synthase domain in red and the membrane in gray. ATP synthase, also called complex V, is the final enzyme in the oxidative phosphorylation pathway. This enzyme is found in all forms of life and functions in the same way in both prokaryotes and eukaryotes. The enzyme uses the energy stored in a proton gradient across a membrane to drive the synthesis of ATP from ADP and phosphate (Pi). Estimates of the number of protons required to synthesize one ATP have ranged from three to four, with some suggesting cells can vary this ratio, to suit different conditions.

This phosphorylation reaction is an equilibrium, which can be shifted by altering the proton-motive force. In the absence of a proton-motive force, the ATP synthase reaction will run from right to left, hydrolyzing ATP and pumping protons out of the matrix across the membrane. However, when the proton-motive force is high, the reaction is forced to run in the opposite direction; it proceeds from left to right, allowing protons to flow down their concentration gradient and turning ADP into ATP. Indeed, in the closely related vacuolar type H+-ATPases, the same reaction is used to acidify cellular compartments, by pumping protons and hydrolysing ATP. ATP synthase is a massive protein complex with a mushroom-like shape. The mammalian enzyme complex contains 16 subunits and has a mass of approximately 600 kilodaltons. The portion embedded within the membrane is called FO and contains a ring of c subunits and the proton channel. The stalk and the ball-shaped headpiece is called F1 and is the site of ATP synthesis. The ball-shaped complex at the end of the F1 portion contains six proteins of two different kinds (three α subunits and three β subunits), whereas the "stalk" consists of one protein: the γ subunit, with the tip of the stalk extending into the ball of α and β subunits. Both the α and β subunits bind nucleotides, but only the β subunits catalyze the ATP synthesis reaction. Reaching along the side of the F1 portion and back into the membrane is a long rod-like subunit that anchors the α and β subunits into the base of the enzyme. As protons cross the membrane through the channel in the base of ATP synthase, the FO proton-driven motor rotates. Rotation might be caused by changes in the ionization of amino acids in the ring of c subunits causing electrostatic interactions that propel the ring of c subunits past the proton channel. This rotating ring in turn drives the rotation of the central axle (the γ subunit stalk) within the α and β subunits. The α and β subunits are prevented from rotating themselves by the side-arm, which acts as a stator. This movement of the tip of the γ subunit within the ball of α and β subunits provides the energy for the active sites in the β subunits to undergo a cycle of movements that produces and then releases ATP.

Mechanism of ATP synthase. ATP is shown in red, ADP and phosphate in pink and the rotating γ subunit in black. This ATP synthesis reaction is called the binding change mechanism and involves the active site of a β subunit cycling between three states. In the "open" state, ADP and phosphate enter the active site (shown in brown in the diagram). The protein then closes up around the molecules and binds them loosely – the "loose" state (shown in red). The enzyme then changes shape again and forces these molecules together, with the active site in the resulting "tight" state (shown in pink) binding the newly produced ATP molecule with very high affinity. Finally, the active site cycles back to the open state, releasing ATP and binding more ADP and phosphate, ready for the next cycle. In some bacteria and archaea, ATP synthesis is driven by the movement of sodium ions through the cell membrane, rather than the movement of protons. Archaea such as Methanococcus also contain the A1Ao synthase, a form of the enzyme that contains additional proteins with little similarity in sequence to other bacterial and eukaryotic ATP synthase subunits. It is possible that, in some species, the A1Ao form of the enzyme is a specialized sodium-driven ATP synthase, but this might not be true in all cases.

Reactive oxygen species Molecular oxygen is an ideal terminal electron acceptor because it is a strong oxidizing agent. The reduction of oxygen does involve potentially harmful intermediates. Although the transfer of four electrons and four protons reduces oxygen to water, which is harmless, transfer of one or two electrons produces superoxide or peroxide anions, which are dangerously reactive.

These reactive oxygen species and their reaction products, such as the hydroxyl radical, are very harmful to cells, as they oxidize proteins and cause mutations in DNA. This cellular damage might contribute to disease and is proposed as one cause of aging. The cytochrome c oxidase complex is highly efficient at reducing oxygen to water, and it releases very few partly reduced intermediates; however small amounts of superoxide anion and peroxide are produced by the electron transport chain. Particularly important is the reduction of coenzyme Q in complex III, as a highly reactive ubisemiquinone free radical is formed as an intermediate in the Q cycle. This unstable species can lead to electron "leakage" when electrons transfer directly to oxygen, forming superoxide. As the production of reactive oxygen species by these proton-pumping complexes is greatest at high membrane potentials, it has been proposed that mitochondria regulate their activity to maintain the membrane potential within a narrow range that balances ATP production against oxidant generation. For instance, oxidants can activate uncoupling proteins that reduce membrane potential. To counteract these reactive oxygen species, cells contain numerous antioxidant systems, including antioxidant vitamins such as vitamin C and vitamin E, and antioxidant enzymes such as superoxide dismutase, catalase, and peroxidases, which detoxify the reactive species, limiting damage to the cell.

Inhibitors There are several well-known drugs and toxins that inhibit oxidative phosphorylation. Although any one of these toxins inhibits only one enzyme in the electron transport chain, inhibition of any step in this process will halt the rest of the process. For example, if oligomycin inhibits ATP synthase, protons cannot pass back into the mitochondrion. As a result, the proton pumps are unable to operate, as the gradient becomes too strong for them to overcome. NADH is then no longer oxidized and the citric acid cycle ceases to operate because the concentration of NAD+ falls below the concentration that these enzymes can use. Compounds Cyanide Carbon monoxide Azide

Use

Effect on oxidative phosphorylation Inhibit the electron transport chain by binding more Poisons strongly than oxygen to the Fe–Cu center in cytochrome c oxidase, preventing the reduction of oxygen. Inhibits ATP synthase by blocking the flow of protons Oligomycin Antibiotic through the Fo subunit. Ionophores that disrupt the proton gradient by carrying CCCP protons across a membrane. This ionophore uncouples Poisons 2,4-Dinitrophenol proton pumping from ATP synthesis because it carries protons across the inner mitochondrial membrane. Prevents the transfer of electrons from complex I to Rotenone Pesticide ubiquinone by blocking to the ubiquinone-binding site.

Malonate and oxaloacetate

Competitive inhibitors of succinate dehydrogenase (complex II).

Not all inhibitors of oxidative phosphorylation are toxins. In brown adipose tissue, regulated proton channels called uncoupling proteins can uncouple respiration from ATP synthesis. This rapid respiration produces heat, and is particularly important as a way of maintaining body temperature for hibernating animals, although these proteins may also have a more general function in cells' responses to stress.

History The field of oxidative phosphorylation began with the report in 1906 by Arthur Harden of a vital role for phosphate in cellular fermentation, but initially only sugar phosphates were known to be involved. However, in the early 1940s, the link between the oxidation of sugars and the generation of ATP was firmly established by Herman Kalckar, confirming the central role of ATP in energy transfer that had been proposed by Fritz Albert Lipmann in 1941. Later, in 1949, Morris Friedkin and Albert L. Lehninger proved that the coenzyme NADH linked metabolic pathways such as the citric acid cycle and the synthesis of ATP. For another twenty years, the mechanism by which ATP is generated remained mysterious, with scientists searching for an elusive "high-energy intermediate" that would link oxidation and phosphorylation reactions. This puzzle was solved by Peter D. Mitchell with the publication of the chemiosmotic theory in 1961. At first, this proposal was highly controversial, but it was slowly accepted and Mitchell was awarded a Nobel prize in 1978. Subsequent research concentrated on purifying and characterizing the enzymes involved, with major contributions being made by David E. Green on the complexes of the electron-transport chain, as well as Efraim Racker on the ATP synthase. A critical step towards solving the mechanism of the ATP synthase was provided by Paul D. Boyer, by his development in 1973 of the "binding change" mechanism, followed by his radical proposal of rotational catalysis in 1982. More recent work has included structural studies on the enzymes involved in oxidative phosphorylation by John E. Walker, with Walker and Boyer being awarded a Nobel Prize in 1997.