MB ChB Year 1: Nutrition and Energy
ATP is a very common metabolite and is present in high concentration (~6mM) in most cells.
People sometimes get the idea that ATP is very rare and precious. In reality, there is stacks of it, and with four negative charges it makes a significant contribution to the total anion content of each cell. ATP was quickly discovered by the early biochemists because there is plenty of it around and it is difficult to miss.
ATP is often described as a “high energy” compound, but hydrolysis of ATP to ADP or AMP yields only modest amounts of energy. Although small, this additional energy tips the balance and is sufficient to drive many unfavourable processes in the required direction.
In general, living cells avoid big changes in free energy, because enzymes are only held together by weak hydrogen bonds and hydrophobic interactions, and it is difficult to build them strong enough to withstand the thump from a violent reaction. The energy released from ATP hydrolysis is not much bigger than the peaks of thermal energy that are constantly rattling molecules around at body heat. But for reactions that are already close to equilibrium, the extra input from ATP is still enough to bias the process in the desired direction. Rather than one big heave at infrequent intervals, living cells use ATP to apply an endless series of little nudges at every point where the flow needs a boost.
ATP carries small packets of energy from place to place. It is the energy currency of the cell. If glucose molecules were £5 notes, then ATP is small change.
Allowing for a few leaks and overheads, complete oxidation of a molecule of glucose would yield about 25 - 30 molecules of ATP and the energy recovery would be about 50%. You may see slightly larger figures in some textbooks, but I think these are a bit optimistic. If glucose is worth a fiver, then each ATP is worth about 10p
There is roughly 75g of ATP in the average human. A reasonably active person (12MJ diet) turns over about 75kg of ATP every day, so a typical ATP molecule is broken down and resynthesised 1000 times each day. In rapidly metabolising tissues the lifetime of each ATP molecule is only a few seconds.
Calculating the lifetime of ATP is a bit misleading. The ratio of ATP:ADP in the cytosol is typically 200:1 or more, and it is the lifetime of ADP rather than ATP which is critical for success. When running for your life, what matters is the maximum possible reaction flux. Diffusion rate is proportional to concentration, so the real bottleneck is returning the "empties" for recycling. The minuscule amount of ADP in working muscle has only milliseconds to get back from the contractile proteins to the mitochondria for reconversion into ATP.
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Muscle contraction apparently requires a very high ATP/ADP ratio, and it is difficult to maintain a low myofibrillar ADP because it will not diffuse quickly enough back to the mitochondria at low ADP levels. (Diffusion rate is directly proportional to concentration.) To overcome this problem, active muscles contain creatine phosphokinase, and large amounts of creatine and creatine phosphate. These compounds equilibrate with the adenine nucleotide pools, and provide high concentrations of two highly diffusible energy carriers to increase the energy transport rate:
About half the total energy available from food oxidation is “captured” in the form of ATP. Of this ATP, about half the free energy can be converted into useful work, such as muscle contraction. Engineering / industrial processes such as electricity generation and motor cars achieve comparable overall efficiencies.
There is a trade off between speed and efficiency, and speed normally wins. In theory, a cell's energy pathways could achieve almost 100% efficiency, providing that they ran infinitely slowly. In the meantime, some more profligate animal has caught up and eaten you. On the other hand, wasteful animals will get very hot and require excesssive amounts of food, so 50% efficiency may be the optimal compromise.
Under aerobic conditions 95% of the cellular ATP is produced within the mitochondria, which actively scavenge the cytosol for ADP, exporting ATP in exchange.
Not only is the mitochondrial ATP factory much more efficient than the other parts of the cell, but all the major energy-yielding pathways are concentrated within the mitochondria.
Mitochondria can really 'Hoover' the place clean, so that it is difficult to detect any free ADP in a eukaryotic cell. There is a fair amount of structural ADP permanently bound onto the cytoskeletal protein actin, but that is not available for use. As we will see in the next lecture, mitochondrial respiration is normally restricted by shortage of ADP.
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The energy available from splitting ATP depends on how far the hydrolysis reaction is displaced from equilibrium. The lower the ADP concentration, the more energy is available. Eukaryotic cells increase the energy yield from each molecule of ATP by transporting ATP and ADP between cell compartments.
The mathematical relationship between DG and the distance from equilibrium was first described by the 19th century American chemist J. Willard Gibbs. It applies to all chemical reactions (in fact, to all physical processes) and in essence is little more than common sense. If you begin with large amounts of reactants and no products then the reaction will proceed to equilibrium with formation of products, but conversely, starting with all products and no reactants the reaction will proceed in the opposite direction to the same final equilibrium position, where DG is zero.
Standard DG0 values are measured with everything at 1 molar concentration, but the actual concentrations in living cells will differ from this, and the effective DG values may vary in different parts of the cell. The precise relationship is a logarithmic one, as shown in the figure below. For different chemical reactions each graph will be displaced along the horizontal axis, but it will always have the same general shape.
Eukaryotic cells do not, however, get something for nothing by pumping ATP from the mitochondria into the cytosol. The transport of ATP, ADP and phosphate across the inner mitochondrial membrane costs them 33% additional energy, over the bare minimum required for the synthesis of ATP within the mitochondrial compartment. This extra energy must be paid for by oxidising additional food, and supplied by the mitochondrial respiratory chain.
Mitochondria have two membranes, which are very different. The outer membrane is smooth and highly permeable. It contains porin, an integral membrane protein that self-assembles into “grommets” with a central hole. Molecules under 5000 daltons have free passage. There are no ionic or electrical gradients across the outer membrane.
The inner membrane thrown into folds called cristae which increase the surface area. It is selectively permeable to small molecules. About a dozen metabolites can cross the inner membrane using highly specific protein carriers, but all other movements are blocked. It has a unique lipid composition which is an excellent electrical insulator. There are very large ionic and electrical gradients across the inner membrane, which are exploited for the synthesis and export of ATP.
Mitochondria from actively respiring tissues like cardiac muscle have a larger internal membrane area that mitochondria from tissues (like liver) with a lower rate of oxygen consumption.
Identify the following key features on this electron microscope picture of a chick embryo mitochondrion:
One of the special phospholipids in the mitochondrial inner membrane is called cardiolipin, or disphosphatidyl glycerol. It has a large fixed negative charge and four fatty acid side chains, instead of the usual two. These provide a very stable anchor, and prevent the membrane pulling to pieces under the huge electrical stress. Barth syndrome is rare genetic problem, affecting only male children, where there is a defect in cardiolipin synthesis. These mitochondria leak, and the children lack energy and develop dilated cardiomyopathy. There is no cure at present, but research continues.
The interior of the mitochondria is called the matrix space. It contains Krebs cycle enzymes, and the later stages of carbohydrate, fat and amino acid breakdown. In liver (but not in other tissues) it also contains part of the urea cycle, and the ancillary enzymes needed to transfer material between the major divisions of metabolism.
Acetyl-CoA produced from either fats or carbohydrates is completely oxidised to carbon dioxide and water by the Krebs (citric acid) cycle in the mitochondrial matrix (see Nelson & Cox pages 571-584). These reactions generate 3 molecules of NADH and one of FADH all of which are re-cycled back to form NAD and FAD by the respiratory chain in the inner mitochondrial membrane. The respiratory chain is composed of flavoproteins, ubiquinone, non-haem iron proteins and cytochromes. [see next lecture]. It conveys electrons stepwise from good reducing agents at the NADH end to good oxidising agents at the oxygen end. The energy released from this process is captured and used for the manufacture of ATP.
Point (don't click) at this image with your mouse to view additional information about the Krebs cycle. We don't expect you to rote-learn this material, but we do expect you to recognise the main compounds, and appreciate their close relationship to key intermediates in amino-acid, carbohydrate and fat metabolism.
The Krebs cycle and the respiratory chain form the final common pathway for substrate oxidation in eukaryotic cells, and the system generates about 95% of the cell's ATP supply. In addition the Krebs cycle is the central clearing house for intermediary metabolism, and provides the essential interface between amino acids, carbohydrates and fats.
The inner membrane has more protein than lipid. It contains numerous respiratory enzymes used for oxygen uptake, proteins involved in metabolite transport and proteins required for the manufacture and export of ATP.
The inner and outer mitochondrial membranes differ enormously in their chemical composition and biological functions.
The intermembrane space between the inner and the outer membranes contains cytochrome c. It also contains creatine kinase and nucleoside diphosphate kinases which share out the energy from ATP and make it available to the remainder of the cell.
Mitochondria cooperate with other organelles in order to achieve the complete breakdown of foodstuffs to CO2 and water. The initial stages of fatty acid activation are performed in the cytosol, and complex lipids are degraded in peroxisomes before the fragments are passed to the mitochondria for final processing.
Most of the cellular lipids contain linear C16 and C18 fatty acids which are activated in the cytosol, and then transfered to the mitochondria as acyl-carnitine derivatives. Fat oxidation within the mitochondria is coupled to the synthesis of ATP.
Very rarely deficiency of CPT1 or CPT2 causes non-ketotic hypoglycaemia in children. This can sometimes be treated by feeding short- or medium-chain length fatty acids, which can enter the mitochondria without using the carnitine shuttle.
Once inside the mitochondria, acyl-CoA is degraded stepwise using β-oxidation to acetyl CoA fragments, producing large amounts of NADH and FADH2. β-oxidation is also known as the lipid oxidation "spiral" because the fatty acid backbone is shortened by two carbon atoms each time the reaction sequence is repeated until eventually there is nothing left. (see Nelson & Cox, page 603-606) Long chain fatty acids (>C12) are processed by a system in the mitochondrial inner membrane, but when the chain length has been reduced to about 12 carbon atoms the partially degraded lipid joins the stream from the peroxisomes and is processed by a soluble enzyme system located in the mitochondrial matrix. Acetyl-CoA from all three sources is finally oxidised via the Krebs cycle within the mitochondrial matrix space.
Fat metabolism nicely illustrates the metabolic specialisation and cooperation between compartments. Fat and steroid biosynthesis takes place mainly in the cytosol, using acetyl-CoA derived from citrate cleavage, although subsequent steroid and bile acid processing occurs in the mitochondrial matrix space. Lipid catabolism is split at least three ways. Xenobiotics, drugs and really awkward molecules get a preliminary free radical assault from the cytochrome p450 system anchored in the endoplasmic reticulum, which introduces additional hydroxyl groups and opens up new lines of chemical attack. The dicarboxylic products of these reactions, together with branched chain fatty acids and very long chain linear fatty acids (>C20) transfer to the peroxisomes, where they are converted to coenzyme A derivatives which undergo a modified type of β-oxidation to reduce them to a manageable size.
Although the peroxisomal fat oxidation "spiral" looks similar to the mitochondrial pathway, it is not connected to the respiratory chain and it does not directly generate any ATP. The first enzyme, acyl-CoA oxidase, is a flavoprotein oxidase that converts oxygen to hydrogen peroxide. The peroxide is detoxified by catalase, which has very high activity in peroxisomes. Hydration and dehydrogenation of the enoyl-CoA are catalysed by a single enzyme, which produces NADH. Peroxisomes lack a respiratory chain, and they are impermeable to pyridine nucleotides so the only available method to recycle the NADH back to NAD is through lactate dehydrogenase, as shown in the diagram below. This is a simple example of a shuttle system (see below) which are widely used to balance up metabolic pathways in different compartments.
Peroxisomal oxidation stops at 8 carbon atoms, so the octanoyl-CoA and acetyl-CoA products of peroxisomal lipid breakdown are converted to the corresponding carnitine derivatives and shuttled across to the mitochondria to complete their conversion to carbon dioxide and water.
There has recently been great research interest in the various fat oxidation pathways, and in the control of gene expression by the peroxisome proliferator activated receptors (PPARs). This system is intimately connected with the development of obesity and type 2 diabetes, which are major medical problems in the modern world.
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Subcellular compartments increase the controllability and efficiency of eukaryotic cells by segregating key metabolites and keeping related processes together. Conditions within each compartment are separately optimised for the task in hand. Toxic or reactive metabolites are kept away from fragile components.
Almost all eukaryotic cells have multiple internal compartments.
What is the most obvious exception?
Make a list of sub-cellular compartments: cytosol, mitochondria ...
There are SEVEN main compartments without counting the internal subdivisions within the organelles. If you were to include all these plus the various types of transport and storage vesicles there would be about twenty separate spaces. Compartmentation increases metabolic efficiency by bringing substrates and enzymes closer together. It also provides opportunities for control. The cytosol is normally the largest compartment, except for adipocytes which are dominated by a huge globule of fat. In most tissues mitochondria are the second largest space (up to 40% of cell volume in cardiac muscle) and they play a central role in metabolism.
Metabolic processes are precisely divided between the different compartments, and even when the same reaction is observed in two separate spaces, they are found to serve different functions. There is also great specialisation between the various tissues, which we will study in the later lectures.
Small organic anions are readily transported between subcellular compartments. Most of the membrane transport proteins catalyse electroneutral exchanges with other anions or hydroxide ions, so the resulting anion distribution is largely determined by the intracellular pH gradients.
Mitochondrial metabolite carriers: (see Nelson & Cox, pages 684 - 685) The existence of huge electrical and chemical concentration gradients between the cytosol and the mitochondrial matrix space (see next lecture) means that free movement of proteins and small molecules cannot be permitted. Elaborate systems exist for transporting many hundreds of components that travel between the two compartments. Movement of small molecules is catalysed and regulated by membrane transport proteins located in the inner mitochondrial membrane. These porters are highly specific for particular charged species, and often catalyse an exchange between two molecules such that their charges exactly balance. These are known as electroneutral porters. If the charges do not balance we have an electrical exchange.
Electroneutral and electrical carriers differ hugely in their transport properties and we will return to this topic in subsequent lectures. Membrane carrier systems are needed whenever a metabolic pathway crosses from one compartment to another, and they are often expressed in a tissue-specific pattern. Examine the transport stoichiometry and functions of the mitochondrial carriers tabulated above and try to decide which category they belong to.
Coenzymes do not move easily between cell compartments. This allows cells to keep their cytosol more oxidising than their mitochondria, which suppresses lactate production under aerobic conditions. There are very few coenzyme transporters, and elaborate metabolite shuttle networks are used instead to move material from one compartment to another.
The diagram below shows the malate - aspartate cycle, which normally accomplishes the re-oxidation of the NADH generated in the cytosol during aerobic glycolysis. It is necessary because neither NAD nor NADH can directly cross the mitochondrial inner membrane. This cycle must revolve twice for every molecule of glucose that completes the glycolytic pathway.
In general, oxidative energy-yielding processes are concentrated within the mitochondria and use NAD+/NADH coenzymes, while reductive biosynthetic processes are mainly located in the cytosol and utilise NADP+/NADPH coenzymes. Both types of coenzyme are found in all cell compartments. Throughout the cell, the NAD+/NADH pair is largely in the oxidised form, whereas the NADP+/NADPH couple is almost entirely reduced.
NAD and NADP both have the same standard redox potentials, which is calculated with equal amounts of the oxidised and reduced forms. The actual redox potentials inside living cells are very different, because cells actively keep these ratios a long way from 1:1 There is a redox pump inside the mitochondria called the energy linked transhydrogenase which actively transfers hydride ions from NADH to NADP+ using energy derived from mitochondrial respiration. The difference in effective redox potential between the two coenzymes is roughly equivalent to the energy stored in ATP.
Mitochondria have their own circular DNA, RNA and protein synthesis, which are all built on bacterial rather than eukaryotic lines. They have 70S rather than 80s ribosomes. They are susceptible to some anti-bacterial drugs. Mitochondrial ATP synthesis is almost identical to the bacterial system. This suggests that mitochondria are descended from captured bacteria that were enslaved by our eukaryotic ancestors – the endosymbiont hypothesis.
The genetic code used by mitochondria is very slightly different to the nuclear version.
The mitochondrial chromosome is very small, but there are hundreds of copies in a typical cell. Mitochondrial DNA is subject to maternal inheritance. Nucleic acid processing is less reliable in mitochondria and it is a mystery how these copies are normally kept in synchrony. Mitochondrial mutations cause serious diseases.
Maternal inheritance arises because the sperm is so much smaller than the egg, so on fertilisation any mitochondria contributed by the sperm are completely swamped by the thousands of copies in the egg. It is claimed that all the mitochondria in modern humans derive from an African lady "mitochondrial Eve" who lived about 200,000 years ago. The full story may be more complicated than this.
Mitochondrial DNA mutations may affect only part of the body, a condition known as "heteroplasmy". One possible explanation is that the mutation occured during cleavage of the embryo, with the result that only part of the body, or a restricted group of tissues received the mutated stock.
Almost all the ancestral bacterial genes were long since “ripped” from the mitochondria and copied into nuclear DNA where they are easier to control. Most mitochondrial proteins are synthesised on cytosolic ribosomes, and laboriously imported across the outer and the inner mitochondrial membranes. Other eukaryotic organelles may have been acquired in a similar fashion, from other free-living precursors that joined the eukaryotic federation.
Importing most mitochondrial proteins from the cytosol is a lot of trouble for our cells. Proteins must be correctly targetted for the matrix space, inner membrane, inter-membrane space or outer membrane. Protein import is expensive energetically, but it is nevertheless worthwhile because of the better opportunities for regulation that exist in the nuclear genome.
The only protein coding genes remaining on the mitochondrial chromosome specify sticky hydrophobic proteins at the core of the mitochondrial inner membrane. These integral membrane proteins spontaneously insert into the first phospholipid bilayer they encounter, so the only safe place to express them is in the interior of the mitochondrial matrix space, where there is limited opportunity to get it wrong.
In humans, the mitochondrial chromosome has only 13 protein-coding genes which specify a few of the core subunits from respiratory complexes I, III and IV, and part of ATP synthase. In addition, it codes for ribosomal RNA and tRNAs, but the DNA and RNA polymerases, and all the ribosomal proteins are imported from the cytosol!
The mitochondrial organisation is destroyed in the membrane permeability transition, which is a key event during apoptosis, or programmed cell death. Various membrane components are reorganised to form a large pore which permits the escape of cytochrome c. This is a key signaling event in the apoptotic cascade, which is used to destroy tumour cells and invading viruses, and to reshape the body during embryogenesis and growth.
Apoptosis or programmed cell death is an ancient strategy for killing defective or unwanted cells which is probably present in all multicellular organisms, both animals and plants. It is employed extensively within the immune system to select the most useful clones. Apoptosis also plays a major role in embryology, pathology and in cancer chemotherapy. (These references are included for general interest, to illustrate the current direction of research activity. You don't need all this detail for ICU3!) Apotosis is a major anti-cancer and anti-viral mechanism in humans, and it is also involved in growth and differentiation, heart attacks and stroke.
There are at least four independent trigger mechanisms:
Serious DNA damage, sensed by P53 (DNA binding protein)
Free radical damage to cells, sensed by mitochondria.
FAS ligand, TNF-a signalling, sensed by cell surface receptors.
Lytic granule release by cytotoxic cells, e.g. natural killer cells
Apoptosis is opposed by the Bcl-2 oncogene [Bcl = B cell lymphoma] and promoted by Bax.
Mitochondrial permeability transition
Release of cytochrome c from the inter-membrane space
Apaf-1, formation of apoptosome
Caspases: activation of the proteolytic cascade
Inactivation of PARP (poly ADP ribose polymerase) normally required for DNA repair
DNA destruction and cell death
Intracellular apoptotic signalling exploits amplifying cascades of cysteine proteases called caspases that activate their protein substrates by cleaving them after aspartate residues. A major event in some of these signalling pathways is the mitochondrial permeability transition, which involves a bizarre change in mitochondrial compartmentation. The adenine nucleotide carrier and other proteins are incorporated into new transmembrane pores. This creates holes in both mitochondrial membranes, collapsing the inner membrane potential, preventing ATP synthesis and releasing cytochrome c from the inter-membrane space into the cytosol. Once released, the cytochrome c associates with another protein called Apaf-1 and initiates a proteolytic cascade. The ultimate result is fragmentation of the DNA and the orderly dissolution of the cell.
Please do not try to memorise every detail in this!
PARP stands for poly(ADP-ribose) polymerase, an enzyme which ADP-ribosylates a wide variety of nuclear proteins, using NAD as the ADP-ribosyl donor. (You will encounter this curious process in other parts of the cell when you study the actions of cholera and diphtheria toxins on mammalian cells.) Nuclear ADP-ribosylation is strongly induced by the presence of DNA strand breaks. It plays a role in DNA repair and the recovery of cells from moderate DNA damage. Inactivation of PARP implies that the cell has "given up" on DNA repair, and the cellular machinery now regards death as inevitable.
The life or death decision depends on a continuously shifting balance between pro- and anti-apoptotic factors. Radiation and cytotoxic drugs are effective against susceptible tumours because these cells are already teetering on the brink of self-destruction, and the slightest push will tilt the balance. The proto-oncogene Bcl-2 is an anti-apoptotic gene whose protein product inhibits the mitochondrial permeability transition. It is named after B cell lymphomas, which are tumours of the MALT tissue in the small intestine where redundant B cells fail to die at the appropriate time.
There is cross-talk between the different pathways, so (for example) granzymes and caspase 8 can both activate the mitochondrial route, and are consequently subject to modulation by Bcl-2. Once triggered, apoptosis is auto-catalytic because activated caspase 3 can itself activate caspase 8, which can in turn initiate the mitochondrial permeability transition and the release of cytochrome c. The cross-talk and positive feedback normally ensures a clear decision on whether each cell will live or die.
Oxygen is a powerful oxidant, which readily generates free radicals that damage many biological molecules. It can be a fiercely toxic gas, and pre-capillary sphincters normally regulate oxygen delivery to the tissues. Mitochondria have a great avidity for oxygen, and the free oxygen concentration inside most cells is usually kept very low. Red cells have no mitochondria but are constantly exposed to oxygen. Despite elaborate measures to protect themselves from oxidation, they become irreversibly damaged and have to be discarded after about 4 months.
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