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Bioc 460 - Dr. Miesfeld Spring 2008 Bioenergetics and Metabolism Supplemental Reading Key Concepts - Energy Conversion in Biological Systems • Review of thermodynamic principles • The adenylate system is used for short term energy storage - Overview of Metabolic Pathways • Metabolic pathways consist of linked enzymatic reactions • The six major groups of metabolic pathways in nature KEY CONCEPT QUESTIONS IN BIOENERGETICS AND METABOLISM: How is energy from the sun converted to chemical energy? What is reaction coupling and why is it important in metabolic pathways? Biochemical Applications of Bioenergetics and Metabolism: Under anaerobic conditions, many microorganisms can metabolize pyruvate to lactate or to ethanol and CO2. The bacterial strain Streptococcus cremoris is used commercially to make cheese by producing large amounts of lactate from pyruvate, whereas the brewers yeast Saccharomyces cerevisea, is used to produce carbonated beer from germinated barley seeds. ENERGY CONVERSION IN BIOLOGICAL SYSTEMS Figure 1. Biochemically speaking, an organism at equilibrium with the environment is no longer alive. For example, the concentration of glucose is much higher inside cells of a saguaro cactus than it is in the surrounding desert (figure 1). Similarly, the concentration of cellular sodium chloride is lower inside a humpback whale than it is in the surrounding ocean. However, when an organism dies, the intracellular concentration of water, essential ions, and macromolecules, quickly become equilibrated with the surroundings. To put off this inevitable event as long as possible, a living organism must be able to extract energy from the surroundings to maintain a steadystate condition (homeostasis) that is far from equilibrium. To accomplish this task, organisms utilize sunlight and materials from the environment to interconvert energy in the forms of work and heat. 1 of 9 pages Bioc 460 - Dr. Miesfeld Spring 2008 We can think of this energy conversion or transduction in terms of 1) chemical work in the form of macromolecular biosynthesis of organic molecules, 2) osmotic work to maintain a concentration of intracellular salts and organic molecules that is different than the extracellular milieu, and 3) mechanical work in the form of flagellar rotation or muscle contraction. Indeed the cycling of resources and waste between the environment and a living cell provides the necessary materials for this energy conversion. As shown in figure 2, solar energy ultimately provides the energy source for life on earth through a three step process. Figure 2. • Review of thermodynamic principles Bioenergetics is a term that is used to describe energy conversion in biological systems and it incorporates the idea that cells represent an open system that freely exchange energy and matter with the surroundings. To better understand bioenergetics in quantitative terms we need to review three thermodynamic principles; 1) First Law of Thermodynamics, 2) Second Law of Thermodynamics, and 3) Gibbs Free Energy. First Law of Thermodynamics The First Law of Thermodynamics states that energy cannot be created or destroyed, only converted from one form to another. As a demonstration of this principle, the French scientist Antoine Laurent Lavoisier in 1783 used a small chamber holding a guinea pig to measure the heat production of metabolism by determining how much ice melted as a result of respiration (figure 3). Figure 3. The energy potential of a compound can be determined using a "bomb" calorimeter to measure heat transfer as a result of combustion in pure oxygen (O2). For example, the combustion of 1 gram of glucose (C6H12O6) produces carbon dioxide (CO2), H2O and heat. The temperature increase of the surrounding water is a measurement of the amount of energy stored in the glucose. 2 of 9 pages Bioc 460 - Dr. Miesfeld Spring 2008 We can write this equation as: C6H12O6 + 6 O2 6 CO2 + 6 H2O + heat Where heat = q = ΔE = 3.75 ºC/kilogram of water Figure 4. The unit of energy in this example is called a Calorie (kcal) which was originally defined by the amount of heat energy required to raise 1 kilogram of water from 14.5 ºC to 15.5 ºC. This can also be expressed in the international unit of measurement the Joule (J) in which 1 Calorie = 1 kcal = 4.184 kJ. Note that in nutritional sciences calorie with a capital "C" actually refers to a kcal. As first demonstrated by Lavoisier, and consistent with the First Law of Thermodynamics, the total energy potential of this 1 gram of glucose is the same regardless of the metabolic path taken. As illustrated in figure 4, the same amount of energy (15.7 kJ) can be extracted from 1 gram of glucose independent of the path taken. In the case of metabolism, a portion of the energy content of glucose is captured in the form of ATP and the rest is lost as heat. In a steady state condition, there is no net gain or loss of ATP, so all of the energy derived from glucose is eventually lost as heat. It is this metabolic heat that Lavoisier measured in his experiment with the guinea pig. Figure 5. Second Law of Thermodynamics The Second Law of Thermodynamics states that all natural processes in the Universe tend towards disorder (randomness) in the absence of energy input. A live cell is highly ordered as compared to the surroundings and thus energy is required to restrain the natural tendency toward disorder. This concept of disorder is defined by the term entropy (S). Ice melting at room temperature cannot be reversed without the input of energy in the form of electricity to lower the temperature. Once the water is frozen the continued input of electricity restrains the ice crystals from melting (figure 5). Similarly, the metabolic energy required for sustaining life restrains the natural tendency of the molecules to become disordered. Cellular life requires solar energy and conversion of chemical energy into work to restrain entropy. 3 of 9 pages Bioc 460 - Dr. Miesfeld Spring 2008 Gibbs Free Energy The change in free energy (ΔG) between a reacting system under standard conditions (reactants initially at 1.0 M, the pressure is 1 atmosphere and the temperature is 298 K = 25ºC), and the same system once the reaction reaches equilibrium, is defined as the change in standard free energy, ΔGº. The ΔGº value is a measure of the spontaneity of a reaction A B as determined empirically by measuring the concentration of reactants at equilibrium using the equation: ΔG° = - RT • lnKeq (where Keq = [B]b / [A]a as described in lecture 2; "a" and "b" are moles) A reaction where ΔGº = 0 is reversible, and by definition, at equilibrium with the surroundings. A reaction with ΔGº < 0 is highly favorable and referred to as exergonic, or work producing, whereas, a reaction with a ΔGº > 0 is less favorable or endergonic. Biochemists use a slightly different term for the change in standard free energy of a reaction as denoted by, ΔGº’. The standard reaction conditions needed to determine ΔGº’ are the same as those described above for ΔGº except that the pH of the reaction is 7.0 ([H+] is 10-7M) and the concentration of water is 55.5 M. In metabolism, ΔGº’ values come into play in three important ways: 1. Reactions with ΔGº’ << 0 are a driving force to make unfavorable reactions more favorable through the use of shared intermediates (product of reaction 1 is the substrate for reaction 2). Importantly, the ΔGº’ of a coupled reaction is the sum of the ΔGº’ values for each individual reaction. If the conversion of A to B is unfavorable (ΔGº’ > 0), but B is quickly converted to C (ΔGº’ << 0), then the conversion of A to B occurs because the net reaction is favorable as shown below: A B B C A C ΔGº’ = +4 kJ/mol ΔGº’ = -10 kJ/mol ΔGº’ = -6 kJ/mol 2. The free energy released from ATP hydrolysis, which is relatively large (ΔGº’ = - 30.5 kJ/mol), can also be used drive unfavorable reactions. In fact, the first step in glycolysis is catalyzed by the enzyme hexokinase which utilizes ATP hydrolysis to drive the unfavorable reaction of glucose phosphorylation in a coupled reaction as shown below: Glucose + Pi glucose 6-phosphate + H2O ATP + H2O ADP + Pi Glucose + ATP glucose 6-phosphate + ADP ΔGº’ = +13.8 kJ/mol ΔGº’ = -30.5 kJ/mol ΔGº’ = -16.7 kJ/mol 3. The actual change in free energy (ΔG) of the reaction A B is the sum of the change in standard free energy ΔGº’ and the term RT • ln[B]actual/[A]actual, in which the concentration values of A and B are those present in the cell under steady-state conditions: ΔG = ΔGº’+ RT • ln [B]actual / [A]actual The ratio of the product and reactant concentrations under actual conditions in the cell is called the mass action ratio = [B]actual/[A]actual, and needs to be distinguished from the equilibrium constant, Keq = [B]equilibrium/[A]equilibrium, which is the ratio of product and substrate concentrations at 4 of 9 pages Bioc 460 - Dr. Miesfeld Spring 2008 equilibrium. Remember, you don’t want metabolic reactions to reach equilibrium with the surroundings, because once they do, you are no longer alive. • The adenylate system is used for short term energy storage Energy obtained from photosynthesis and oxidation of metabolic fuels drives an ATP synthesis reaction that captures redox energy in the form of phosphoanhydride bond energy. Importantly, this bond energy can be readily recovered by ATP cleavage and used to drive chemical, osmotic and mechanical work. The highest energy form is ATP which contains two phosphoanhydride bonds as shown in figure 6. Figure 6. As shown in figure 7, the breakdown of macromolecules through catabolic pathways yields ATP, whereas, anabolic pathways used to synthesize macromolecules in the cell require ATP hydrolysis to drive unfavorable reactions. The term adenylate system refers to the interconversion of low and high energy forms of adenylate between ATP, ADP, and AMP. To see why the adenylate system is important consider that a 70 kg person requires ~100 moles of ATP every day based on the energy content of food, assuming that ~40% of the potential energy released from metabolism is converted to ATP. The molecular weight of ATP is 507 g/mol, which means we hydrolyze as much as 50 kg of ATP every day! Rather than synthesizing our own weight in ATP on a daily basis, it is much more efficient to recycle adenylate forms by reforming ATP from ADP + Pi. This is done in two ways. Figure 7. Since ATP is the high energy form of the adenylate system, then the ratio of the concentration of ATP to the concentration of ADP and AMP in the cell at any given time can be used as a measure of the energy state of the cell. This relationship can be expressed in terms of the Energy Charge (EC) of the cell which takes into account the number of phosphoanhydride bonds available for work: 5 of 9 pages Bioc 460 - Dr. Miesfeld Spring 2008 Most cells are found to have an EC in the range of 0.7 to 0.9 which means that the [ATP] is higher than [ADP] or [AMP]. This can be represented schematically by the graph in figure 8. Energy charge is a useful concept when thinking about metabolism. As shown in figure 9, when EC<0.8, then the activity of phosphorylating systems (catabolism) increases to replenish ATP levels (battery power is low, time to recharge). Conversely, when EC > 0.8, then biosynthetic pathways (anabolism) are more active to take advantage of the high [ATP]. Figure 8. Figure 9. OVERVIEW OF METABOLISM Metabolic pathways consist of a series of reactions that are coupled together through the metabolism of shared intermediates (figure 10). Metabolic pathways can be linked together to form linear pathways, cyclic pathways and branched pathways (figure 11). Figure 10. Figure 11. 6 of 9 pages Bioc 460 - Dr. Miesfeld Spring 2008 The term metabolic flux refers to the rate at which metabolites are degraded and synthesized by a series of linked reactions. For example the metabolic flux through glycolysis is higher than metabolic flux through gluconeogenesis when more glucose is converted to pyruvate (glycolysis) than pyruvate is converted to glucose (gluconeogenesis). Three primary mechanisms control metabolic flux, 1) the amount of rate-limiting enzyme (changes in gene transcription or protein synthesis), 2) tha catalytic activity of rate-limiting enzymes (covalent modifications or allosteric regulation) and 3) bioavailability of substrates (nutritional supplies or cell comparmentalization). One of the best ways to understand how flux through various catabolic and anabolic pathways changes in response to substrate concentration and enzyme activity levels, is to look at glucose metabolism in the liver before and after breakfast as illustrated in figures 12 and 13. Early in the morning before your first meal, blood glucose levels begin to decline after a night of “fasting” which triggers glucagon release from the pancreas. Glucagon signaling in liver cells activates both a catabolic pathway (glycogen degradation) and an anabolic pathway (gluconeogenesis), while at the same time inhibiting the catabolism of glucose by the glycolytic pathway. After breakfast, insulin levels increase due to high blood glucose which stimulates glucose uptake, glycogen synthesis, and glucose catabolism via the glycolytic pathway. Figure 12. Figure 13. The six major groups of metabolic pathways in nature The breakfast scenario gives the take-home message for the rest of the lectures in this course; metabolic pathways are highly interdependent and exquisitely controlled by substrate availability and enzyme activity levels. Even though we examine one pathway at a time for pedagogical purposes, the key to understanding metabolic integration in terms of nutrition, exercise, and disease (e.g., diabetes and obesity) is learning how metabolic flux between pathways is controlled. 7 of 9 pages Bioc 460 - Dr. Miesfeld Spring 2008 Figure 14. To try and keep things interesting (and organized), we will approach the next 16 lectures as a journey through a metabolic forest (i.e., the proverbial don't lose sight of the forest through the trees). As with any extended trip, we need three items for our journey, 1) a good map to figure out where we are, 2) an itinerary to keep on schedule, and 3) a guidebook to point out the important landmarks. Figure 14 shows the metabolic map we will use to keep track of the pathways (download a full page copy from the study guide page on the website). This metabolic map illustrates the hierarchical nature of metabolism which includes four classes of macromolecules (proteins, nucleic acids, carbohydrates, and lipids), six primary metabolites (amino acids, nucleotides, fatty acids, glucose, pyruvate, acetyl CoA) and six small biomolecules (NH4+, CO2, NADH, O2, ATP, H2O). This metabolic map will be used as a template each time a pathway is introduced using the “divide and conquer” strategy illustrated in figures 15 and 16. Figure 15. Figure 16. The first group of pathways we will discuss are those involved with the oxidation of monsaccharides through a series of redox reactions culminating in ATP synthesis (glycolysis, citrate cycle, electron transport chain, oxidative phosphorylation, photosynthesis). Lectures 32-40 cover three of the groups of pathways shown in blue that together control the synthesis and degradation of 1) carbohydrates (carbon fixation, pentose phosphate pathway, gluconeogenesis, glycogen synthesis and degradation), 2) lipids (fatty acid synthesis and degradation, lipid 8 of 9 pages Bioc 460 - Dr. Miesfeld Spring 2008 transport, cholesterol and steroid synthesis) and 3) amino acids (including nitrogen metabolism). Nucleotide metabolism is covered in Bioc 461 (or Bioc 411). Finally, in lectures 41 and 42, we will tie the various pathways together by looking at how metabolic integration in humans explains normal (nutrition and exercise) and abnormal (diabetes and obesity) physiological states. We will start off the discussion of each new pathway by answering the following four questions about the pathway: 1. What does the pathway accomplish for the cell? 2. What is the overall net reaction of the pathway? 3. What are the key enzymes in the pathway? 4. What are examples of this pathway in real life? These questions (and more importantly, the answers) function as our “guidebook” by highlighting the contributions of each pathway to the overall metabolism of the cell. Moreover, they will help you review your trip through the various pathways in preparation for exams 3 and 4. ANSWERS TO KEY CONCEPT QUESTIONS: Life on earth is made possible by the biochemical reactions of photosynthesis, carbon fixation and aerobic respiration which together convert solar energy into ATP (and NADPH) which is used to synthesize carbohydrates from CO2 and H2O. Aerobic organisms, such as ourselves, consume carbohydrates as a chemical source of energy and metabolize them in the presence of O2 to from CO2 and H2O. All organisms depend directly or indirectly on energy derived from thermonuclear fusion reactions on the sun to prevent (for as long as possible) reaching equilibrium with the environment; a high entropy state called death. Reaction coupling permits energetically unfavorable reactions to be more favorable in the context of a pathway. Coupling ATP hydrolysis to a phosphoryl transfer reaction is one example of reaction coupling that takes place in the same enzyme active site. The net ΔGº’ for an ATP coupled reaction is often highly negative, for example, the phosphorylation of glucose by the enzyme hexokinase. Another type of reaction coupling is when two enzymes in pathway are energetically linked through a shared common intermediate. Since the actual change in free energy, ΔG, is the sum of the change in standard free energy, ΔGº’, and RT•ln(mass action ratio), in which the mass action ratio is [product]actual/[substrate]actual , depletion of a reaction product by its metabolism as a substrate in the coupled reaction, results in a reduction in ΔG since ln of a mass action ratio <1 is a negative number. Therefore, even though the ΔGº’ for a reaction is a positive number (based on the reaction reaching equilibrium in a test tube under ideal conditions), the actual ΔG is a negative number because RT•ln(mass action ratio) is a negative number due to lower than "expected" [product] in the cell due to its function as a substrate in a linked reaction of the pathway. 9 of 9 pages