1. Energy The meaning of “energy” in energy metabolism In a haste to learn the individual reactions in a pathway, its easy to lose sight of the purpose of the pathway. With energy metabolism, the purpose is to generate energy, generally as ATP or NADH or some high energy compound that will be used in a later anabolic step. Glycolysis and Krebs cycle reactions have a high number of kinase and dehydrogenase enzymes, respectively, for this reason. This class of enzymes is intimately connected with energy production and conservation. Pathways in the cytosol tend to be less energy yielding, whereas those in the mitochondria are almost totally devoted to energy production. This tutorial will bring you closer to understanding why and how cells conserve energy. It will also help you see the logic behind molecular energy calculations. As you listen to your heart pump or move your arm to scratch your head, you should be able to tell what purpose energy serves to life.

2. The terms energy conservation and energy generation tend to carry the same meaning. Conservation implies “avoiding heat”, or channeling the energy differential between reactants and products into the synthesis of a compound. Because energy as heat cannot be exploited in an isothermal system, biological systems have to conserve energy by biosynthesis. Suppose for example ATP is hydrolyzed during a reaction (click 1). The standard energy differential between reactants and products (  G o’ ) of that reaction is 30.5 kJ/mol. What is energy conservation? This means the environment of the cell gains 30.5 kJ of heat energy for each mole of ATP hydrolyzed by water. Obviously, this is wasteful. To counter the loss, ATP hydrolysis is coupled with the synthesis of a phosphorylated compound. You saw this as a “coupled” reaction when ATP was needed to produce glucose-6-PO 4 or fructose 1,6-bisPO 4 (click 1). Now you see that by making glucose-6-PO 4 or fructose 1,6-bisPO 4, the cell avoids losing the larger part of the ATP hydrolysis energy as heat. This is energy conservation. Click one to go on. ATP + H 2 O ADP + PO 4 Glucose + ATP Glucose-6-PO 4 + ADP Fructose-6-PO 4 + ATP Fructose 1,6-bisPO 4 + ADP

3. Direct vs Indirect Energy Production The energy generated in metabolic pathways comes in two forms, direct or indirect. Direct or “substrate level” refers to energy generated during a particular reaction. The production of ATP by reacting ADP with PEP is an example of this type (click 1) COO CH 3 C=O COO CH 2 C~OPO 3 = + ADP + ATP Indirect refers to energy channeled into a compound that will return the energy at a later step. High energy compounds such as acyl-phosphates or thioesters fit this example. Another is NADH generated during oxidation reactions in the cytosol or Krebs cycle. When L-malate is oxidzed by NAD +, NADH is generated (click 1). NADH and FADH 2 have trapped the electron pair from the oxidation in their structures and will release the energy when they themselves are oxidized. COO CH2 HO-C-H COO C=O COO CH2 COO + NAD + + NADH + H + : :

4. Calculating energy yield in glycolysis Calculating energy yield helps you see the energy phase of metabolism in real numbers. Take for example the energy yield when glyceraldehyde-3-PO 4 is oxidized to pyruvate. How much energy is conserved in this reaction? To determine that number we need to know the pathway. We also need to know if anaerobic or aerobic conditions prevail. First the pathway. There are 5 enzyme- catalyzed reactions to consider (click 1). glyceraldehyde-3-PO 4 + PO 4 + NAD + 1,3-bisPO 4 glycerate 3-phosphglycerate 2-phosphoglycerate PEP 2-phosphoglycerate PEP pyruvate + ATP + H 2 O glyceraldehyde-3-PO 4 + PO 4 + NAD + + 2ADP pyruvate + NADH + H + + 2ATP + 2H 2 O Removing the common terms on both sides yields a final equation (click 1). We see that the phosphate on glyceraldehyde-3-PO 4 and the inorganic PO 4 both contribute to formation of ATP. Thus, 2 ATPs are formed by the 5 reactions. Under anaerobic conditions “two” represents the final yield. But, if the reaction was carried out with oxygen and involved the mitochondria, energy to the equivalent of 5 ATPs would result. Click 1 to see why. + NADH + H + 3-phosphoglycerate + ATP + ADP + H 2 O

5. Energy yield in the mitochondria The mitochondria is the heart of aerobic metabolism. Electrons in NADH and FADH 2 are channeled into the electron transport system, which is driven by O 2. A large part of energy of oxidation of the electron transport components is preserved in ATP. Each NADH generates the equivalent of 3 ATPs and each FADH 2, 2 ATPs for each pair of electrons transferred to oxygen (click 1). O2O2 H2OH2O NAD NADHElectron transport ATP NADH from the cytosol yields its electrons indirectly via a shuttle. NADH generated by the 3 NAD-linked dehydrogenases in the Krebs cycle provide most of the energy. For example, each citrate molecule oxidized to CO 2 and H 2 O generates the equivalent of 36 ATPs. Click 1 to see how this value was obtained. :

6. Energy yield in the Krebs cycle A cycle implies the last intermediate returns to the front. Each turn of the Krebs cycle results in the loss of 2 carbons as CO 2 and generates 3NADH, one FADH 2 and one GTP (click 1). A 2-carbon compound, such as the acetate group on acetyl-CoA, therefore, yields 12 ATPs of energy. Acetyl-CoA citrate isocitrate  -ketoglutarate succinyl-CoA succinate fumarate malate oxaloacetate NADH GTP FADH 2 NADH CO 2 Now, suppose instead of acetyl- CoA we want to determine the ATP yield when oxaloacetate (OAA) is oxidized (click 1). First write the equation for the oxidation (click 1). OAA yields 4 moles of CO 2 for each mole oxidized. Thus, 2 turns of the cycle are needed to oxidize all of the carbons in OAA to CO 2. Two turns is equivalent to 24 ATPs. Performing the same analysis for citrate shows 6CO 2 liberated, or 3 turns of the cycle (click 1). Thus, citrate yield 36 ATPs, or one third more energy than OAA. Finally lets consider the oxidation of malate (click 1). Malate has 4 carbons, which means the oxidation will generate 4CO 2. But, we also need to oxidize malate to OAA, which generates one NADH. Thus 3 more ATPs than OAA, i.e., 24 + 3= 27 ATPs. Click 1 to test and expand your understanding. C 4 H 4 O 5 + 3 1/2 O 2 4CO 2 + 2H 2 O C 6 H 8 O 7 + 4 1/2 O 2 6CO 2 + 4H 2 O C 4 H 6 O 5 + 5 O 2 4CO 2 + 3H 2 O

7. Test and Expand your understanding about energy 1. How many phosphorylated intermediates are in the Krebs cycle? Ans: None. GTP is synthesized from GDP + P i. GTP, however, is not a cycle intermediate. 2. How is ATP generated in the Krebs cycle? Ans: Indirectly. The reduced coenzymes, NADH and FADH 2 shuttle electrons to the electron transport system and energy is preserved by ATP synthesis 3. Is pyruvate → acetyl-CoA the only way to enter carbons into the Krebs cycle? Ans: No. Any compound that can be converted into a Krebs cycle intermediate will contribute carbons to the Krebs cycle. This applies to aspartate and glutamate, which form OAA and  -ketoglutarate, respectively. 4. What numbers should I remember in order to calculate energy yield in the Krebs cycle? Ans: In terms of ATP, remember that each NADH is equivalent to 3, each FADH 2 to 2, and each turn of the cycle 12 ATPs. 5. How many ATPs are generated when succinyl-CoA is oxidized in the cycle? Ans: 30. One for GTP, two for FADH 2 and 3 for NADH must be added to the 24 for 2 turns of the cycle.