The Krebs Cycle

- Also known as the tricarboxylic acid cycle (TCA cycle), or the citric acid cycle

- Discovered in 1937 by Sir Hans Krebs, a biochemist working at the University of Sheffield in England

- Eight-step process, each step catalyzed by a specific enzyme; cyclic because oxaloacetate is the product of the last step as well as the reactant in the first step

- Overall chemical equation for the Krebs cycle: oxaloacetate + acetyl-CoA + ADP + Pi + 3NAD⁺ + FAD → CoA + ATP + 3NADH + 3H⁺ + FADH2 + 2CO2 + oxaloacetate


- By the end of the Krebs cycle, the original glucose molecule is entirely consumed. The 6 carbon atoms of the original glucose molecule leave the process as 6 low-energy CO2 molecules. The energy of the glucose is preserved and stored in the form of 4 ATP molecules (2 from glycolysis, 2 from Krebs cycle), and 12 reduced coenzymes (2 NADH from glycolysis, 2 NADH from pyruvate oxidation, 6 NADH from the Krebs cycle, and 2 FADH2 from the Krebs cycle). Most of the free energy stored in NADH and FADH2 will eventually be transferred to ATP through processes called electron transport and chemiosmosis.


Steps of the Krebs Cycle:

1. The acetyl group (2-C) of acetyl-CoA condenses with oxaloacetate (4-C) to form citrate (6-C).
2. Citrate (6-C) is rearranged to isocitrate (6-C).
3. Isocitrate (6-C) converted to a-ketoglutarate (5-C) by losing a CO2 and two hydrogen atoms that reduce NAD⁺ to NADH.
4. a-ketoglutarate (5-C) is converted to succinyl-CoA (4-C). A CO2 is removed, coenzyme A is added, and two hydrogen atoms reduce NAD⁺ to NADH.
5. Succinyl CoA (4-C) is converted to succinate (4-C). ATP is formed by substrate level phosphorylation, and coenzyme A is released.
6. Succinate (4-C) is converted to fumarate (4-C). 2 hydrogen atoms reduce FAD to  FADH2.
7. Fumarate (4-C) is converted to malate (4-C) by the addition of H2O.
8. Malate (4-C) is converted to oxaloacetate (4-C). 2 hydrogen atoms reduce NAD⁺ to NADH. The cycle repeats.

Key Features of the Krebs Cycle:

- Since two acetyl-CoA molecules are formed from one glucose molecule, the cycle occurs twice for each molecule of glucose processed.


- In step 1, acetyl-CoA enters the cycle and reacts with a molecule of oxaloacetate (OAA) to produce a molecule of citrate. In this reaction, OAA (4-C) is converted into citrate (6-C) by adding the 2-C acetyl group of acetyl-CoA, releasing CoA, which is recycled. Also, OAA has 2 carboxyl groups and citrate has 3 carboxyl groups.


- Energy is harvested in steps 3, 4, 5, 6, and 8.


- In steps 3, 4, and 8, NAD⁺ is reduced to NADH.


- In step 5, ATP is formed by substrate-level phosphorylation. A phosphate from the mitochondrial matrix displaces CoA from succinyl-CoA. The phosphate is then transferred to guanosine diphosphate (GDP) to form guanosine triphosphate (GTP). Next, phosphate condenses with ADP, forming ATP. Overall, free energy is transferred from succinyl-CoA to ATP.


- Energy is harvested in step 6. However, reaction is not exergonic enough to reduce NAD⁺ to NADH. Instead, free energy is stored by reducing FAD to FADH2, a step closely related to the electron transport chain in mitochondria.


- Last 4 carbon atoms of the original glucose leave as CO2 in steps 3 and 4 (two Krebs cycles to process 1 glucose). The CO2 molecules diffuse out of the mitochondrion and the cell as metabolic waste.

Enzyme Lab Results

Factor tested: temperature

Trial #	Temperature of Substrate (degrees C)	Initial Volume (mL)	Final Volume (mL)	Change in Volume (mL)	Time (seconds)
1	30	105	230	125	40.9
2	22	230	350	120	82.6
3	10	350	480	130	102.6

Metabolism and the Laws of Thermodynamics

The Three Laws of Thermodynamics and how they apply to metabolism are described below:

1. The Conservation of Energy. The amount of energy in the universe is constant. Energy cannot be created or destroyed but may be converted from one form to another. 

The First Law applies to metabolism in the sense that energy is not free. For example, if the body needs to do a certain amount of work - let's say 5 kJ - the body needs to consume 5 kJ of chemical energy in the form of food to do the 5 kJ of work required. Any energy that is released by an exergonic reaction is absorbed by the surroundings. Conversely, any energy that is stored by an endergonic reaction causes a commensurate decrease in energy of the surroundings.

2. The Law of Entropy. The entropy in an isolated system increases with any changes that occur. All spontaneous events act to increase total entropy.

Entropy is a measure of the randomness or disorder in a collection of objects or energy. Everything in the universe favours an increase in entropy. Therefore, reactions that produce an increase in entropy are favoured over reactions that produce a decrease in entropy, and the metabolic processes in living things are no exception. Living organisms obey the Second Law of Thermodynamics. When they use anabolic processes to make complex ordered structures like proteins and DNA, they are creating order out of chaos. However, these processes must be accompanied by an even greater disorder caused by energy-yielding catabolic processes. For example, a child lifting a potato chip to his mouth results in an increase in gravitational potential energy and a decrease in entropy. The child obtains the necessary free energy for this action through the entropy-producing catabolic reactions of digestion and cellular respiration. In the end, the entropy produced by the metabolic processes is greater than the decrease in entropy produced by moving the potato chip to the mouth, resulting in a net increase in the entropy of the universe. In conclusion, "living organisms create order out of chaos in a local area of the universe at the expense of creating a greater amount of disorder in the universe as a whole."

3. Absolute Zero. Absolute zero is the temperature (-273°C) at which all thermal kinetic energy ceases. Nothing can be colder than absolute zero. 

Metabolism is unable to proceed at extremely low temperatures close to absolute zero because of the fact that all molecular motion ceases, making chemical reaction unable to occur. Also, enzymes are unable to function at extremely high and extremely low temperatures.