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Overview of Metabolism David P. Gardner, Ph.D. M2P Course 2024 Objectives 1. Explain, with respect to energy in the cell, why aldolase B deficiency is much more serious than fructokinase deficiency. 2. Explain why cells do not violate the1st and 2nd laws of thermod...
Overview of Metabolism David P. Gardner, Ph.D. M2P Course 2024 Objectives 1. Explain, with respect to energy in the cell, why aldolase B deficiency is much more serious than fructokinase deficiency. 2. Explain why cells do not violate the1st and 2nd laws of thermodynamics. 3. Identify if a reaction is likely to be spontaneous based upon its change in free energy (𝚫G). 4. Explain the difference between an exergonic reaction and an endergonic one and relate this to 𝚫G. 5. Describe how linking an endergonic reaction to ATP hydrolysis can allow the reaction to go forward. 6. Give an example of how low [ATP] affects more than just enzymes. 7. Relate anabolic and catabolic reactions to storage and mobilization. 8. Explain why fats are more energy dense than carbohydrates. 9. Compare and contrast oxidation and reduction reactions in terms of C-H bonds. 10. Explain the mechanism whereby fatty acid oxidation stimulates gluconeogenesis and explain why this stimulation is important. Recommended Readings Lippincott Biochemistry, 8e Chpt. 6. Images are from Lippincott Biochemistry unless otherwise noted. Disorders of Fructose Metabolism Fructokinase deficiency: autosomal recessive disorder where fructose accumulates in the urine but is otherwise benign. Affects ~1/30,000 live births. Aldolase B deficiency: autosomal recessive disorder where fructose 1-P cannot be converted into glyceraldehyde and dihydroxyacetone phosphate. Affects ~1/20,000 live births. Symptoms: Severe renal hemorrhage hyperuricemia hypoglycemia dysfunction Symptoms are only seen lactic with ingestion of liver failure jaundice death acidemia fructose, sucrose or sorbitol. Fructose Metabolism Aldolase B step is one step further in the pathway than fructokinase. Why so much more severe? Aldolase B deficiency causes a profound ATP deficiency after fructose, sucrose or sorbitol ingestion (sorbitol is converted to fructose by sorbitol dehydrogenase). Fructokinase deficiency does not. Aldolase B Deficiency Fructose enters hepatocytes and is rapidly converted into fructose 1 phosphate by fructokinase. Reaction transfers a phosphate group from ATP to fructose with generation of ADP. Next step is blocked. Phosphate within the cell is ___________ in fructose 1-P. ATP levels rapidly plunge without available supply of inorganic phosphate. Many enzymatic reactions become thermodynamically unfavorable. Why? 1 and 2 st nd Laws of Thermodynamics 1 law: total energy of a system and its surroundings remains constant st Energy can neither be created nor destroyed. 2nd Law: degree of disorder in a system (entropy) increases over time. How do cells increase order over time? Do they violate the 2nd law? Changes in entropy within a cell are balanced by changes in the cellular environment. A reaction that decreases entropy within a cell must generate sufficient heat to _____________ the overall entropy of the surroundings. Importance of ATP: Driving Chemical Reactions The direction and extent of chemical reactions depends upon the free energy of the reaction (G) or Gibbs free energy. The change in free energy (𝚫G) is an indication of the ability to do work. It predicts if a reaction is favorable. At equilibrium 𝚫G is zero. The sign of a 𝚫G can predict the direction of a reaction. Reactions with a negative 𝚫G proceed spontaneously to equilibrium. Reactions with a positive 𝚫G require an input of energy to take place. Chemical Reactions Panel A is a spontaneous reaction. Panel B is not. Compound A has a higher free energy (G) than compound B. If 𝚫G of A → B is -5 kcal/mol, then B → A is +5kcal/mol. A → B is exergonic (releases energy) while B → A is endergonic (requires energy input). 𝚫G also depends upon the concentration of reactants and products. A high [B] could drive the reaction. Enzymes and Chemical Reactions Enzymes have _____ effect on the 𝚫G of a reaction. Enzymes lower the amount of energy that must be put into the high energy intermediate (activation energy). Enzymes make it much easier for substrates to achieve the transition state energy. ATP Hydrolysis and Chemical Reactions For a series of reactions, the 𝚫G is additive. Thus linking a reaction with a high -𝚫G to a reaction with a +𝚫G can allow the unfavorable reaction to go forward. Reactants Products 𝚫G° Glucose + H2PO4 Glucose 6-P + H20 +3.3 kcal/mol ATP +H20 ADP +HPO4-2 -7.3 kcal/mol Glucose + ATP Glucose 6-P + ADP -4.0 kcal/mol 𝚫G° is under standard conditions, not cellular conditions. The overall 𝚫G glucose to glucose 6-P to fructose 6-P is favorable. ATP → ADP + Pi has a 𝚫G° of -7300 cal/mol. In a cell ( 𝚫G) is closer to - 12,000. Many endergonic reactions in cells are linked to ATP hydrolysis to drive them forward. What happens when ATP levels crash? ATP to AMP Pay particular attention to reactions that hydrolyze ATP → AMP. Hydrolysis of ATP to AMP yields similar energy to ATP → ADP. However, hydrolysis to AMP also produces pyrophosphate, which is highly ___________. Its hydrolysis has a 𝚫G similar to ATP → ADP. Thus hydrolysis of ATP to AMP yields approximately 2X the energy. FIGURE 2.6 ATP as a store of free energy Molecular Biology of the Cell, 9th Edition Aldolase B and ATP Deficiency In aldolase B deficiency, depletion of ATP is especially pronounced in the liver where much of dietary sugars are metabolized. 60% of dietary glucose is metabolized by the liver. A deficiency of ATP will have catastrophic effects on the ability of a cell to carry out critical enzyme reactions. Additionally, membrane pumps like the Na+/K+ ATPase will slow and result in inability to maintain osmotic balance. Water moves into the cells resulting in swelling and rupture. FIGURE 14.25 Model for operation of the Na+-K+ pump Molecular Biology of the Cell, 9th Edition Metabolic Pathways Can be divided into anabolic and catabolic pathways. This can often be viewed as storage (anabolic) vs. mobilization (catabolic), especially for lipids and carbohydrates. When glucose is high, we store the glucose for future use. When glucose is low, we ___________ the stores. Generation of sufficient ATP/NADH/ NADPH also critical. Intermediary Metabolism Very useful figure from Lippincott biochemistry textbook. (Fig. 8.2) Key aspects: Pathways are interconnected. Regulated steps are emphasized. Reversible steps are common but are not regulated steps. Enzyme reactions where high energy compounds are either generated or required are key. Catabolic Reactions Hydrolysis of complex molecules to their components e.g. proteins to amino acids. Conversion to acetyl CoA Oxidation reactions to generate ATP. Anabolic Reactions Generation of complex products from more simple substrates. Requires energy input. Often involve oxidation of NADPH to provide high energy electrons. Key steps in fat biosynthesis involve incorporation of high energy electrons of NADPH into the new C- H bonds of the lipid being synthesized. Substrates for Catabolic Reactions _______ are the predominant fuel source during catabolism. Typical 70 kg male: 30 kg muscle, 10 kg fat. Comparison of fuels in 70 kg male at beginning of a fast. Why are fats so energy dense? Palmitic acid 9000 kcal/kg 4000 kcal/kg Catabolic Oxidation of Fatty Acids Fatty acid synthesis involves the synthesis of a highly reduced 16 carbon fatty acid. Fatty acid catabolism involves the oxidation of that fat. The energy released from the oxidation of C-H bonds is captured in either NADH or FADH2. Oxidation of 16C palmitate yields 8 acetyl CoA, 7 NADH and 7 FADH2. What happens to those is a topic for another day. Pathway Interconnection During a fast, nervous tissue is dependent upon blood glucose as a fuel source. Fatty acids cannot be used by neurons. In a fast, both fatty acid oxidation and glucose synthesis (gluconeogenesis) need to increase in the liver. Acetyl CoA is the key positive allosteric regulator of pyruvate carboxylase. Pyruvate carboxylase is the key regulated enzyme of gluconeogenesis. Thus, β-oxidation of fats stimulates gluconeogenesis.