Biology

Key Unity Competence
Describe the structure and importance of ATP, outline the roles of the coenzymes NAD, FAD and coenzyme A during cellular respiration.

Learning objectives
–– Discuss the need for energy in living organisms as illustrated by anabolic reactions, active transport, and the movement and maintenance of body temperature.
–– Describe the structure of ATP as a phosphorylated nucleotide formed by condensation reaction.
–– Explain that ATP is synthesized in substrate-linked reactions in glycolysis and in Krebs (tri-carboxylic acid [TCA] cycle.
–– Explain the relative energy value of carbohydrate, lipid and protein as respiratory substrate and explain why lipids are particularly energy-rich.
–– Define the term Respiratory Quotient (RQ) as the ratio of the volume of CO₂, evolved to the volume of O₂ uptake during aerobic respiration.
–– Design simple experiments using respirometers to determine the RQ of germinating seeds or small invertebrates. Example: woodlice.
–– Calculate RQ values from the equations of respiration of different substrates.
–– Interpret graphs for varying RQ values during seed germination.

This unit deals with the energy from respiration. It focuses on the description of the structure and importance of adenosine triphosphate (ATP), and outline the roles of the coenzymes including nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD) and coenzyme A CoA during cellular respiration. Specifically, this unit contributes to a better understanding of the reasons why organisms need energy, the structure of adenosine triphosphate (ATP), synthesis and breakdown of
ATP, respiratory substrates and their relative energy values, and measurement of respiration and respiratory quotient.

5.1 Need for energy by organisms

Chemical energy is the most important type of energy potential for life, where energy is either released out or consumed through metabolism reactions. Metabolism reactions constitute the sum of all chemical reactions taking place in a living cell. The biological process by which metabolic pathways breakdown molecules into smaller units that are either oxidized to release energy is called catabolism, while the biological process by which a set of metabolic pathways construct molecules from smaller units through reactions consuming energy is called anabolism. During catabolism reactions, energy is released to the surrounding environments. These are exergonic reactions. During anabolism reactions, energy is absorbed from the surrounding environment. These are endergonic reactions.

All living organisms need energy to grow and reproduce, maintain their structures, and respond to their environments. Metabolism reactions are the set of life-sustaining chemical processes that enables organisms to transform the chemical energy stored in molecules into energy that can be used for cellular processes. Animals consume food to replenish energy. Their metabolism breaks down the carbohydrates, lipids, proteins, and nucleic acids to provide chemical energy for these processes. Plants convert light energy from the sun into chemical energy stored in molecules during the process of photosynthesis.

Active transport of solutes such as sodium (Na+), potassium (K+) magnesium (Mg+), calcium (Ca+) and chloride (Cl-)across the plasma membrane cannot be possible without the use of energy. The transport proteins that move solutes against their concentration gradients are all carrier proteins rather than channel proteins. Active transport enables a cell to maintain internal concentrations of small solutes that differ from concentrations in its environment. Some transport proteins act as pumps, moving substances across a membrane against their concentration or electrochemical gradients. Energy is usually supplied by adenosine triphosphate (ATP) hydrolysis (Figure 1).

5.2 Structure of Adenosine Triphosphate and its importance

The special carrier of energy is the molecule of adenosine triphosphate (ATP). The building blocks of ATP are carbon, nitrogen, hydrogen, oxygen, and phosphorus, contained in the ribose sugar, a nitrogen base called adenine and a chain of phosphate group (Figure 5.2)

ATP has the following biological functions in the cell:

a. Active transport

ATP plays a critical role in the transport of macromolecules such as proteins and lipids into and out of the cell membrane. It provides the required energy for active transport mechanisms to carry such molecules against a concentration gradient.

b. Cell signaling

ATP has key functions of both intracellular and extracellular signaling. In nervous system, adenosine triphosphate modulates the neural development, the control of immune systems, and of neuron signaling.

c. Structural maintenance

ATP plays a very important role in preserving the structure of the cell by helping the assembly of the cytoskeletal elements. It also supplies energy to the flagella and chromosomes to maintain their appropriate functioning.

d. Muscle contraction

ATP is critical for the contraction of muscles. It binds to myosin to provide energy and facilitate its binding to actin to form a cross-bridge. Adenosine diphosphate (ADP) and phosphate group (Pi) are then released and a new ATP molecule binds to myosin. This breaks the cross-bridge between myosin and actin filaments, thereby releasing myosin for the next contraction.

e. Synthesis of DNA and RNA

The adenosine from ATP is a building block of RNA and is directly added to RNA molecules during RNA synthesis by RNA polymerases. The removal of pyrophosphate provides the energy required for this reaction. It is also a component of DNA.

Adenosine triphosphate (ATP) is the energy currency for cellular processes. It provides the energy for both energy-consuming endergonic reactions and energy-releasing exergonic reactions. When the chemical bonds within the phosphate group of ATP are broken, energy is released and can be harnessed for cellular work.

The hydrolysis of ATP to ADP and Pi is a reversible reaction, where the reverse reaction combines ADP + Pi to regenerate ATP from ADP. Since the hydrolysis of ATP releases energy, ATP synthesis must require an input of free energy. Recall that free energy is the portion of system’s energy that can perform work when temperature and pressure are uniform throughout the system. In a living cell, ADP is combined with a phosphate group to form ATP in the following biochemical reaction: ADP+ Pi + free energy → ATP+H₂O

b. ATP and energy coupling

Now that the synthesis and breakdown of ATP is understood, the remaining interesting question is to know exactly how much free energy denoted ∆G is released with the hydrolysis of one mole of ATP, and how is that free energy used to do cellular work. The calculated ∆G for the hydrolysis of one mole of ATP into ADP and Pi is estimated at −7.3 kcal/mole equivalent to −30.5 kJ/mol. However, this is only true under standard conditions, and the ∆G for the hydrolysis of one mole of ATP in a living cell is almost double the value at standard conditions and equals -14 kcal/mol or −57 kJ/mol. ATP is a highly unstable molecule. Unless quickly used to perform work, ATP spontaneously dissociates into ADP + P_i, and the free energy released during this process is lost as heat. To harness the energy within the bonds of ATP, cells use a strategy called energy coupling.

5.4 Respiratory substrates and their relative energy values

A respiratory substrate refers to the substance required for cellular respiration to derive energy through oxidation. They include carbohydrates, lipids and proteins.

Carbohydrates include any of the group of organic compounds consisting of carbon, hydrogen and oxygen, usually in the ratio 1:2:1. Hence the general formula of carbohydrates is . The examples of carbohydrates include sugars, starch and cellulose. Carbohydrates are the most abundant of all classes of biomolecules, and glucose whose chemical formula is C₆H₁₂O₆ is the most known and the most abundant. Its breakdown produces energy in the following way:

C₆H12O₆ +6 O₂→6 CO₂ +6 H₂O+Energy   (ATP + heat).

This breakdown is exergonic metabolic reaction, having a free-energy change of -686 kcal (2,870 kJ) per mole of glucose decomposed.

Lipids include diverse group of compounds which are insoluble in water but dissolved readily in other lipids and in organic solvents such as ethanol (alcohol). Lipids mainly fats and oils contain carbon, hydrogen and oxygen, though the proportion of oxygen is lower than in carbohydrates. Fats and oils have a higher proportion of hydrogen than either carbohydrates or proteins. This property makes them a more concentrated source of energy, where each gram of fat or oil yields about 38kJ (38 kJ/g) more than twice the energy yield of a gram of carbohydrate.

Proteins are other respiratory substrate. They are large and complex biological molecules which play many and diverse roles during respiration. They mainly work as enzymes. Enzyme is a biological catalyst that controls biochemical reactions in living organisms

Back to glucose when it is broken down during the process called glycolysis, the dehydrogenases enzymes transfer electrons from substrates, here glucose, to NAD+ which in turn forms NADH. At this stage the electron transport chain accepts electrons from NADH and passes these electrons from one molecule to another in electron chain transfer leading to a controlled release of energy for the synthesis of ATP. At the end of the chain, the electrons are combined with molecular oxygen and hydrogen ions (H+) to form one molecule of water.  (Figure 5). When NAD is oxidized, its oxidized form NAD+ is converted into its reduced from NADH, and two molecules of ATP are produced.

The transformation of succinate to fumarate, the sub-products of the breakdown of glucose during glycolysis process, two hydrogens are transferred to flavin adenine dinucleotide (FAD), forming FADH₂. The reduced coenzymes NADH and FADH₂ transfer higher energy electrons to the electron transport chain. Finally, another coenzyme called coenzyme A sometimes abbreviated by CoA, a sulfur-containing compound is attached via its sulfur atom to the two-carbon intermediate, forming acetyl CoA. The Acetyl CoA has a high potential energy, which is used to transfer the acetyl group to a molecule in the citric acid cycle, a reaction that is therefore highly exergonic producing great number of energy in the form of ATP.

5.5 Measurement of respiration and respiratory quotient

The rate of respiration is measured by the use of respirometer device, typically by measuring oxygen consumed and the carbon dioxide given out. It can also be used to measure the depth and frequency of breathing, and allows the investigation on how factors such as; age, or chemicals can affect the rate of respiration. Currently, the computer technology is also used to automatically measure the volume of gases exchanged and drawing off small samples to analyse the proportions of oxygen and carbon dioxide in the gases

The respiratory quotient (RQ) is the ratio of the volume of carbon dioxide produced to the volume of oxygen used in respiration during the same period of time. The RQ is often assumed to equal the ratio of carbon dioxide expired: oxygen inspired during a given time as it is summarized in the following formula:

The RQ is important as it can indicate whether the respiration is aerobic or anaerobic.

C₆H₁₂O6 +6 O₂→6 CO₂ +6 H₂O+ Energy (ATP + heat).

As each molecule of gas occupies the same volume, this would give RQ = 1.0, and this is common for all carbohydrates. Further studies indicated the respiratory quotient to be 0.9 for proteins and 0.7 for fats, and concluded that an, RQ greater than 1.0 indicates anaerobic respiration, while RQ equals or less than 1.0 indicates aerobic respiration.

Note that respiration during germination, especially in early stages was also studied. Results indicated that it is difficult for oxygen to penetrate the seed coat, so that at this stage, the RQ is about 3 to 4. Later when the seed coat is shed, it becomes easier for oxygen to reach respiration tissues and the levels of RQ falls. Results indicated that eventually seeds with large carbohydrate stores have an RQ around 1.0 and those with large lipid stores have RQs of 0.7 to 0.8.

This graph suggests that the seed begins with carbohydrate as a metabolite, changes to fat/oil then returns to mainly using carbohydrate

a. Measuring and obtaining the RQ values during seed germination process

During seed germination, CO₂ is released. To test its presence, chemicals including Sodium hydroxide or Potassium hydroxide are used due to their ability to absorb CO₂. As the germinating seeds use oxygen, pressure reduces in tube A so the manometer level nearest to the seeds rises (figure 5.8). The syringe is used to return the manometer fluid levels to normal. The volume of oxygen used is calculated by measuring the volume of gas needed from the syringe to return the levels to the original values. If water replaces the sodium hydroxide, then the carbon dioxide evolved can be measured.

Measuring and obtaining the RQ values in invertebrate (e.g. woodlice)

In this particular respirometer, woodlice have been placed in a boiling tube which is connected to a U-tube. The U-tube acts as a manometer (a device for measuring pressure changes). The other end of the U-tube is connected to a control tube which is treated in exactly the same way as the first tube, except that it has no woodlice but instead glass beads which take up the same volume as the woodlice. The two boiling tubes (but not the manometer) are kept in water bath at constant temperature. The U-tube contains a coloured liquid which moves according to the pressure exerted on it by the gases in the two boiling tubes. Both tubes contain potassium hydroxide solution which absorbs any carbon dioxide produced.

When the woodlice respire aerobically, they consume oxygen, which causes the liquid to move in the U- tube in the direction of arrows. The rate of oxygen consumption can be estimated by timing how long it takes for the liquid to rise through a certain height. The experiment can be repeated by replacing the potassium hydroxide solution with water. Comparing the changes in manometer liquid level with and without potassium hydroxide solution gives an estimate of carbon dioxide production can be used to measure the respiratory quotient.

If the internal radius of the manometer tube is known, the volumes of gases can be calculated using the equation:

Volume of gases = π r² h,

where π is equal to 3.14, r is the internal radius of the tube and h is the distance moved by the liquid.