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Self-Instructional on Glycolysis and Respiration: The Production of ATP from Glucose

How Organisms Convert Food to Energy

Do you ever wonder how organisms convert food to energy?

We all know that organisms must take in nutrients to survive. This self-instructional unit describes two of the most important processes in food to energy conversion. These processes, called glycolysis and respiration, use the energy of the glucose molecule to manufacture ATP, the most widely used energy molecule of the cell.

First let's discuss the structure and function of ATP.

ATP is an abbreviation for the nucleotide Adenosine triphosphate. This nucleotide consists of a ribose, a phosphate and the nitrogenous base adenine. This is the same nucleotide used to manufacture RNA. Don't be surprised by the fact that ATP is very energetic. All of the nucleotides used to make RNA and DNA are full of energy. Most of their energy is stored in the two bonds between their two terminal phosphates. The nucleotides of DNA are called deoxyribonucleotides (abbreviated dATP, dTTP, dCTP and dGTP).  The nucleotides of RNA are called ribonucleotides (abbreviated ATP, UTP, CTP, and GTP). While these nucleotides have different types of ribose sugar and nitrogenous bases, they all have the two terminal phosphate groups, and so they all have equivalent energies.  All the energy in the nucleotide comes from the two terminal phosphates.

The energy used to make DNA and RNA polymers, through the formation of phosphodiester bonds, is supplied by the triphosphate nucleotides when they polymerize to form nucleic acids.

It takes energy to form all chemical bonds, and the breaking of these chemical bonds will release energy. The triphosphate nucleotides are special because the bonds between the phosphates take more energy to form (~8 kilocalories/mole) as compared to the bonds between the phosphate and the ribose (~5 kilocalories/mole). Therefore these high energy bonds between the phosphates release more energy when they are broken.

Figure 1. The structure of ATP

The phosphates in the structural formula of ATP have been assigned specific labels based on the IUPAC system. The phosphate attached to the #5 carbon of the ribose is labeled as the alpha (a) phosphate. The next two phosphates are labeled beta () and gamma (g) from left to right. The bonds between the beta and gamma phosphates are represented by wavy lines which indicate the higher energy potential of these bonds.

Most biochemical reactions require energy input to proceed. These types of reactions are called endergonic reactions. An exergonic reaction, by contrast, gives off energy and proceeds spontaneously. Many chemical reactions of the cell use the energy from ATP which released when the beta or gamma phosphate bonds of ATP are broken. The energy released from the ATP supplies the energy necessary to form or break chemical bonds in biochemical reactions. The energy released by the conversion of ATP to ADP is used in the manufacture of ATP during the process of glycolysis.

When the phosphates are cleaved from ATP, two other forms of the nucleotide are made; ADP (adenosine diphosphate) and AMP (adenosine monophosphate). If you cover the terminal (gamma) phosphate in the structural formula of ATP with a piece of paper you will see what ADP looks like and if you cover the gamma and beta phosphates you will see what AMP looks like.

ATP is made inside cells by the transfer of a phosphate on to the b phosphate of ADP. Where does ADP come from? ADP is always inside cells as the byproduct of the cleavage of a gamma phosphate from ATP. This is the ultimate use of recycling by the cell!  Sometimes the ATP, ADP and AMP molecules are completely degraded because they wear out. Cells contain many biochemical pathways and can rebuild new AMP molecules from scratch when necessary. The AMP molecule can then be phosphorylated to form ADP and ATP. However, the cell prefers to conserve energy, whenever possible, by the dephosphorylation of ATP to ADP and AMP and the rephosphorylation of these back to ATP. The following reaction represents the phosphorylation and dephosphorylation reactions of ATP.

Figure 2.

Practice Round One

1. Draw a diagram of ATP and label the significant energy-containing bonds.

2. Where is the energy stored in the ATP molecule?

3. How do AMP and ADP differ from ATP?

4. Define exergonic and endergonic reactions.

How Cells Use Glucose for the Manufacture of ATP

The first step of the breakdown of glucose inside the cell, to utilize the potential energy stored in this molecule, is a process called glycolysis. Any basic college biology text will have all of the steps of the process of glycolysis. In this self instructional I want to give you an overview of the process, in the hope that you will understand what is taking place and not just memorize the process.

Where does glycolysis take place?

Glycolysis takes place inside the cytoplasm of the cell. The cytoplasm is the aqueous based solution inside a cell that has a variety of molecules solubilized in it. The contents of the cytoplasm include proteins, carbohydrates, nucleic acids, salts and a whole host of other soluble molecules.

What are the parts of the cell that are not considered cytoplasm?

The organelles inside the cytoplasm are not part of the cytoplasm. Remember, most organelles are bound by lipid membranes and are not soluble in the cytoplasm. The contents of organelles are also isolated from the cytoplasm due to the hydrophobic nature of their membranes. This means that the substances solubilized inside organelles stay separate from the rest of the cytoplasm. The enzymes necessary for glycolysis are solubilized in the cytoplasm of the cell.

What does the process of glycolysis accomplish?

Figure 3. Splitting of glucose through glycolysis

Why are only two net ATP produced during glycolysis?

Although glycolysis allows for the formation of four ATP, two ATP are used in the process. This results in a net profit of two ATP from the process of Glycolysis.

Does glycolysis require molecular oxygen? No, glycolysis is an anaerobic process.


Practice Round Two

1. What does the process of glycolysis accomplish in terms of ATP manufacture and end product formation?

2. Where does glycolysis occur and what are the destinations in the cell for its end products?

3. What does the cytoplasm of a cell contain?

4.Where does the term glycolysis get its name?

Enzymes and Enzymatic reactions

To help understand the processes of glucose manufacture, anabolic reactions, and catabolic reactions, we need to understand how enzymes work.

Enzymes are a group of biological molecules, most of which are made of proteins. Enzymes are biological catalysts. A catalyst is added to a chemical reaction to speed the rate of the reaction. In other words, a catalyst makes the reaction occur faster. The catalyst is not changed in its form, but instead retains its original physical and chemical characteristics at the completion of the reaction. A catalyst does not participate in a reaction and can be reused again and again. Enzymes have all the qualities of a catalyst, but they are specifically used in the chemical reactions of living organisms (metabolism). Often people say; enzymes break this substance down or form this substance; however, to state the function of the enzyme correctly, one should say enzymes catalyze the formation or breakdown of a substance.

How do enzymes work? Enzymatic catalysts reduce the amount of energy necessary for endergonic reactions to proceed. Enzymes don't supply the energy, they reduce the amount needed. How? Well, there are several theories concerning this.

One way to speed reactions is to concentrate the materials that participate in them. Remember the generalized formula for chemical reactions?

Reactant A + Reactant B -------> Product C + Product D

Enzymes are molecules that have a 3-dimensional shape. Think of a muffin tin with all the depressions used to hold the individual muffins. If one hole contains reactant A and the adjacent hole contains reactant B, the two reactants are in close proximity to one another. This has the effect of concentrating the two reactants necessary for an anabolic reaction to occur and form products C and D.

Remember the cytoplasm of the cell or even the contents of an organelle may seem tiny, but it is a vast ocean of many components to a glucose or other small molecule.

Concentrating reactants is one way to speed reactions. Another way to reduce the energy and speed the rate of catabolic reactions might be to hold a molecule in place so that some of its bonds are under strain. Imagine you were trying to cut a thick piece of cloth with dull scissors and the going was very slow. If two people were to hold the material taut, or under strain, the fabric would cut more easily. Perhaps enzymes hold molecules in a similar fashion allowing bonds to be broken more easily.

If enzymes only speed the rates of reactions, does this mean that chemical reactions occur spontaneously anyway? Yes, all chemical reactions can occur spontaneously, but some reactions may take millennia to occur. Since organisms do not live on a geological time scale, the ability of enzymes to speed the rate of reactions, up to thousands of times faster, is necessary for life to exist.

The Structure of Enzymes

Enzymes are 3-dimensional molecules that have folded up into a specific shape (eg. the muffin tin). Imagine the depression in our muffin tin to be the site at which the chemical reaction takes place. This site is called the active site of the enzyme. The molecule that fits in the active site of the enzyme is called the substrate. Enzymes and substrates are very specific for each other. The active site of an enzyme is designed to fit perfectly with its specific substrate, like a 3-dimensional jigsaw puzzle.

Biochemical or enzymatic pathways

Sometimes a cell needs a product from a reactant that requires only a single chemical reaction. For example, reactant A needs to be changed to product B. This reaction would only require the enzyme that fits with its substrate, reactant A. However, this is not usually the case. Most products inside cells that need to be broken down or built up require a multi-step process. This process is called a biochemical pathway. It goes something like this:

Finally a quick lesson in naming enzymes. Generally to name an enzyme we take the substrate it breaks down or product that it manufactures and add an -ase on the end. Examples: enzymes that degrade the following substances DNAse, ATPase or enzymes that build up the following substances: DNA polymerase and ATP synthetase.

Practice Round Three

1. List and define the characteristics of a catalyst.

2. How do enzymes assist biochemical reactions to occur ?

3. How do enzymes and their substrates interact?

4. Define anabolism, catabolism and metabolism.

5. How do we name enzymes?

Glucose Utilization

The figure below demonstrates some of the possible pathways for glucose utilization by a variety of organisms.

Figure 3. Pyruvate metabolism in different organisms

Figure 4. Glycolysis as a Biochemical Pathway

Please note that the conversion of glucose to pyruvate requires 10 enzyme reactions and occurs in 10 separate steps. After glycolysis, what happens to the pyruvate molecules?

Both of the pyruvate (three-carbon molecules) are immediately converted to two-carbon molecules by removing a CO2 (carbon dioxide) from each molecule. The remaining two-carbon compound is known as an acetyl group. The acetyl group will react quickly with another molecule because of its unpaired electrons. Therefore as the CO2 is cleaved from pyruvate, the acetyl group is added simultaneously to a carrier molecule called coenzyme A (CoA).

Coenzyme A (CoA) is not an enzyme but a molecule similar in some respects to a nucleotide. This is a very important carrier molecule because it carries the acetyl group to many important biochemical pathways. The acetyl group and CoA molecule combine to form acetyl CoA. This molecule travels to the matrix of the mitochondria.

Once there, the CoA transfers the two-carbon acetyl group to an enzyme which catalyzes the addition of a four-carbon compound called oxaloacetic acid (oxaloacetate) to the acetyl. The result of this reaction is a six-carbon compound called citric acid (citrate). This is why this biochemical pathway is called the citric acid cycle.

The citric acid molecule has carbon, hydrogen, and oxygen atoms stripped from it in a step-wise fashion through this biochemical pathway. Each step requires its own specific enzyme. The end result of this pathway is the regeneration of oxaloacetic acid, and the conversion of NAD and FAD to NADH and FADH with the formation of carbon dioxide CO2 as a byproduct.

The major objective of the citric acid cycle is to get the hydrogens, along with their electrons, off the carbon compounds so they can be carried to the electron transport proteins that are embedded in the membrane of the mitochondrion.

The waste or by-products of these reactions are the carbon and oxygen that are stripped off as each compound is rearranged by the removal of hydrogen. These carbons and oxygens combine to form CO2 carbon dioxide gas which is given off as a byproduct of these reactions. The CO2 that you breathe out of your lungs is a waste product coming from the cellular metabolism of glucose!

Practice Round Four

1. What happens to pyruvate from the end of glycolysis to the end of the citric acid cycle?

2. If the citric acid cycle both begins and ends with oxaloacetic acid what is the main objective of this process and how does it occur?

Figure 5. A simplified diagram of the citric acid (Krebs) cycle.

Carbon dioxide (CO2) enters our bloodstream, eventually winds up in our lungs, and is expelled upon exhalation, but where do those hydrogens with their electrons go? The hydrogens and electrons are picked up by electron carrier NAD (nicotinamide adenine dinucleotide) and electron carrier FAD (flavin adenine dinucleotide). These electron carriers transfer the hydrogens and electrons to the electron carrying proteins, embedded in the mitochondrial membrane, which form the electron transport chain. The electron transport chain proteins are complexed with metal ions which allow them to carry extra electrons safely.

When NADH and FADH arrive at the electron transport chain, they transfer the electrons associated with the hydrogens to the electron carrying proteins at the top of the electron chain.

Figure 6. The structural formula for NAD and NADH.

When electrons and hydrogen atoms are added to a molecule, this is called reduction. When electrons or hydrogens are removed from a molecule, this is known as oxidation. NAD is in an oxidized state while NADH is in a reduced state.

Now that the electrons have been transferred to the electron transport proteins, the hydrogen ion H+ (proton) is free to be pumped across the membrane into a space between the inner and outer mitochondrial membranes.

This proton pumping requires energy which is generated by the flow of electrons as they are passed down the electron transport protein chain. As electrons are transferred from one protein to the next, energy is released at each transfer, until the final electron acceptor, oxygen is reached.  Aerobic organisms must take in oxygen because it serves as the final electron acceptor in the electron transport chain. 

The energy, released from the passage of the electrons down the electron transport chain to oxygen, is used to pump the protons across the membrane. The pumping of protons requires a great deal of energy, because they have to be pumped against a concentration gradient.

Because protons are constantly being pumped across the membrane, they fill the space between the mitochondrial membranes. This is known as an area of high proton concentration. Putting more protons in this area would be like trying to force balloons into a box already filled to capacity with balloons.

Pushing protons against a concentration gradient of protons is referred to as going against, or up, a concentration gradient. In the case of the balloons, as soon as you open the box they would come out easily with no energy input necessary. This is termed going with, or down, the concentration gradient. Molecules naturally flow from areas of high concentration to areas of low concentration with no energy input necessary.

Once you have captured all this energy by pushing protons up the proton concentration gradient into a crowded area, what happens? The protons can get out but they only have one passage to escape. This is a passage created by a protein enzyme called ATP synthetase. The ATP synthetase phosphorylates (adds phosphates to) ADP. The ATP synthetase gets the energy necessary for the formation of the high energy phosphate bond from the rush of protons through the channel it creates across the mitochondrial membrane.

To better understand how the flow of protons provides energy for the ATP synthetase enzyme imagine how a turbine engine works at Hoover Dam. The rushing water is caught and causes the huge turbine to turn which generates electricity. In a similar way the energy of protons cascading through the ATP synthetase is converted into the energy used to form the phosphate bond of ATP.

This process, called oxidative phosphorylation, makes most of the ATP manufactured by cells. While glycolysis gives us net profit of two ATP, the electron transport coupled with the citric acid cycle and oxidative phosphorylation give us a net profit of approximately 34 ATP from each glucose catabolized. Quite a big difference in yield!

What happens to the protons and electrons when they flow back through the membrane and down electron transport chain? Since electrons are energetic enough to cause problems inside the cell they must be inactivated. The final electron acceptor, oxygen, combines with the electrons and excess hydrogen ions to form water, a non-toxic by-product.

So the products of respiration are CO2 (from the citric acid cycle) and H2O (from electron transport) and lots of ATP. Without oxygen, aerobic cells die because the manufacture of ATP is halted at the cellular level. Since oxygen is absolutely necessary for the continual phosphorylation of ADP to ATP this process is called oxidative phosphorylation. Without oxygen, electron transport would stop, protons would not be pumped, and the ATP synthetase would not have a flow of protons to supply the energy necessary for the manufacture of ATP.

Do you know where all the molecules come from and their destination in the following Generalized Equation for Respiration?

C6H12O6 + O2  ------>  CO2 + H2O + energy

Now I would like to give you my overview of all of the processes of glucose metabolism in narrative form. I hope that you will understand the processes of glycolysis and respiration well enough to be able to describe this process in a similar way. In other words, I would like you to be able to think through and understand the concepts, not the minute details of these processes.