Background Information

Enzymes are an important class of proteins that function as catalysts in living cells. You may have already used EnzymeLab to learn about basic principles of enzyme activity and the role of enzymes in metabolism. Cell metabolism is dependent on the combined actions of many different enzymes that are essential for the anabolic and catabolic reactions that must occur to maintain the physiology of a cell. It is important to understand that many reactions that require enzymes rarely involve just a single enzyme that works by an all-or-none process. This would be like trying to manufacture a car from all of its components in one sweeping motion! Instead many biochemical reactions involve cascades of enzymatic reactions called metabolic pathways. In a metabolic pathway, several enzymes work in a sequential fashion to convert reactions into a product or products. At each step in a metabolic pathway, intermediate molecules (metabolites) are produced that serve as the substrates for subsequent enzymes in the pathway. One benefit of a metabolic pathway compared to a single-enzyme reaction is that a cell can often precisely regulate the amount of product generated by independently controlling the catalytic activity of certain enzymes in the pathway.

Intermediate molecules are often an important part of the control of a metabolic pathway. One way in which metabolic pathways can be regulated by intermediates involves a process called allosteric regulation. In this form of control, a molecule binds to a portion of an enzyme, other than the active site, that alters the shape of the active site to either stimulate or inhibit the enzyme. Feedback inhibition is another mechanism that cells can use to regulate a metabolic pathway. In feedback inhibition, a final or end-product of a reaction can inhibit enzymes in the metabolic pathway. This process allows a cell to carefully control the amount of end-product that it produces as a way to prevent excess accumulation and waste of an end-product. Frequently, the end-product inhibits or blocks the activity of an enzyme at one of the initial, rate-limiting steps in the pathway to prevent the unnecessary production of intermediates. This would be similar to a car manufacturer, whose car supply has exceeded the public's demand, stopping his auto assembly line at the first step rather than halfway through the assembly process to avoid producing half-completed cars.

One of the most essential metabolic pathways that occur in all living cells involves the enzymatic conversion of food molecules to produce energy in the form of a molecule called adenosine triphosphate (ATP). ATP is a critical energy source universally utilized by all living cells. ATP powers many chemical reactions by providing phosphate groups that are necessary for phosphorylation reactions. Phosphorylation reactions are catalyzed by a broad class of enzymes called kinases. Kinases can transfer a phosphate group from ATP onto other molecules. Phosphorylation of molecules such as proteins, often results in a change in the shape of the phosphorylated protein that activates the protein to perform a desired function. For example, muscle contraction requires that the contractile proteins in a muscle cell, actin and myosin, are phosphorylated thus enabling these two proteins to slide along one another to produce shortening of the muscle cell. Although lipids and proteins can undergo catabolism in cells to serve as metabolic fuels to power the reactions necessary for ATP synthesis, carbohydrates such as glucose are excellent sources of energy for producing ATP. Many plant and animal cells, including human cells, can convert glucose into ATP in the presence of oxygen by a set of reactions called aerobic cellular respiration. Other cells -- for example certain yeast cells, bacteria, and human skeletal muscle cells -- can produce ATP from glucose in the absence of oxygen (anaerobic conditions) via reactions called fermentation. Cell respiration produces a much higher total yield of ATP from one molecule of glucose than fermentation does while producing a minimal number of waste products. In addition to ATP, other end-products produced include water and carbon dioxide, the primary waste product produced by cell respiration. The summary equation showing the net yield of products created by the catabolism of one molecule of glucose (C6H12O6) is shown below:

C6H12O6 + 6 O2 -> 6 H2O + 6 CO2 + 36 ATP

Glucose catabolism via cellular respiration can be grouped into three major metabolic stages, these are (1) glycolysis, (2) the Krebs cycle also known as the citric acid cycle, and the tricarboxylic acid cycle (TCA cycle), and (3) the electron transport chain and oxidative phosphorylation. In eukaryotic cells, glycolysis occurs in the cytoplasm of the cell. The Krebs cycle occurs in the mitochondrial matrix while the reactions of the electron transport chain and oxidative phosphorylation occur on the cristae of the mitochondrion. These pathways rely on oxidation reduction reactions in which electrons are enzymatically removed (oxidation) from glucose and transferred (reduction) to electron acceptor molecules such as nicotinamide adenine dinucleotide (NAD+). Upon receiving electrons, NAD+ is reduced to NADH which functions as an electron carrier that supplies electrons to an electron transport chain in mitochondria that will ultimately power ATP synthesis in the reactions known as oxidative phosphorylation. The reactions of cellular respiration are summarized in the next three paragraphs.

Glycolysis involves ten enzymatic steps that ultimately result in the degradation of one molecule of glucose into two molecules of a three-carbon acid called pyruvate (pyruvic acid). Glycolysis also results in the net production of two ATP molecules and two molecules of NADH. These reactions can occur under aerobic or anaerobic conditions but the yield of pyruvate and NADH is always the same. Before entering the Krebs cycle, each pyruvate is subsequently converted into a molecule called acetyl coenzyme A (acetyl CoA).

The Krebs cycle involves eight enzymatic steps that serve to oxidize both molecules of acetyl CoA with the primary purpose being the production of six molecules of NADH and two molecules of an electron carrier molecule similar to NADH called flavin adenine dinucleotide (FADH2). These reactions were originally known as the tricarboxylic acid cycle or citric acid cycle because each enzymatic step results in the production of eight organic acids called carboxylic acids (citrate, isocitrate, a-ketoglutarate, succinyl CoA, succinate, fumarate, malate, and oxaloacetate) as intermediate molecules. Each of these molecules serves as a substrate for the next enzyme in the Krebs cycle and some of these intermediates serve to regulate enzymes of cell respiration. You will be designing experiments with some of these intermediates using MitochondriaLab to test the effects of each intermediate on the reactions of cellular respiration.

During the reactions of the electron transport chain, the electrons stored in the NADH and FADH2 produced by the Krebs cycle are enzymatically transferred to a series of electron acceptor molecules in the cristae. Many of the electron acceptor molecules are iron-containing proteins called cytochromes. The cytochromes are clustered together within the cristae to form four multiprotein groups or complexes of proteins named groups I, II, III, and IV, that transfer electrons in a sequential fashion. Molecular oxygen serves as the final electron acceptor in this chain. Oxygen molecules receive electrons from complex IV. This reaction represents the oxygen-consuming stage of cellular respiration because reduced oxygen molecules are converted into water during this transfer. As electrons are being transferred along this chain, this series of oxidation-reduction reactions generates a H+ gradient (called a proton-motive force) in the intermembrane space of the mitochondrion. This H+ gradient provides the energy necessary for an enzyme called ATP synthase. This enzyme also functions as an ion channel to allow H+ flow down a gradient from the intermembrane space into the mitochondrial matrix. The flow of H+ through ATP synthase activates the enzyme to synthesize ATP from ADP and inorganic phosphate in a final stage called oxidative phosphorylation.

Studying the reactions of cellular respiration is an excellent way to develop an understanding of the complexities of cell metabolism and the mechanisms by which a cell can control its metabolism. One way to study the reactions of cell respiration involves performing experiment on preparations of mitochondria that have been isolated from mitochondria-rich tissue, such as skeletal muscle or liver, using cell fractionation techniques. Many of the details of these reactions have been well understood for almost 100 years. Numerous scientists were involved in carrying out the biochemical experiments that were used to learn about the substrates, enzymes, and cofactors required for cellular respiration, the sequence of each reaction relative to one another, and the regulation of these reactions. These reactions were some of the first well-characterized and well-understood examples of a metabolic pathway.

Some of the landmark experiments that led to our understanding of these reactions involved classic techniques in biochemistry that employed glass reaction flasks into which diced tissue preparations, intermediates, end-products, waste products, and inhibitors could be added independently, in combination, or in sequence. Biochemical activities such as the production of end-products, consumption of intermediates, and rates of enzyme activity were then measured to follow the rates of the reaction. Modern-day biochemists continue to rely on experiments of this design to study metabolism.

Many investigators contributed to research in the late 1800s and early 1900s that unraveled the sequence of reactions that occur during glycolysis. Biochemical studies in the early 1900s demonstrated that preparations of diced animal tissues exhibited the enzymatic ability to carry out oxidation reduction reactions by transferring hydrogen ions from acids such as citrate, malate and fumarate to dye molecules that would change color when they were reduced. Albert Szent-Gyorgi carried out some of the earliest studies. When performed under aerobic conditions his studies established that organic acids could be oxidized to produce carbon dioxide and water, and that inhibitors of these reactions blocked the consumption of oxygen in the reaction.

Another pioneer of cellular respiration was the German biochemist Hans Krebs who discovered the citric acid cycle that now more commonly bears his name as the Krebs cycle. Krebs proposed that this pathway involved a cycle of enzymatic reactions that were required to oxidize organic acids such as pyruvate. Many of Krebs' experiments involved studying the oxidation of organic acids in slices of flight muscle from pigeons. To support active wing flapping over long distances, these muscle cells depend heavily on oxidative metabolism to produce ATP so they served as an excellent tissue source for Krebs' research. Krebs also determined that the use of inhibitors such as malonate could block the production of some intermediates in the cycle while causing the accumulation of other intermediates. Using this approach, Krebs was able to determine the sequence of enzyme steps involved in the citric acid cycle by comparing what intermediates were consumed and which ones were accumulated following the addition of inhibitors. The importance of Krebs' work was rightfully acknowledged when he was awarded the Nobel Prize in 1953 together with Fritz Lipmann who discovered a coenzyme that is an important electron carrier molecule involved in the electron transport chain.

In the 1920s and 1930s many scientists worked independently to determine the steps involved in electron transport. In 1961, British biochemist Peter Mitchell first proposed a mechanism called chemiosmosis as the process during oxidative phosphorylation that requires a hydrogen ion gradient for the production of ATP. Mitchell carried out most of his research using bacteria. As was the case with Hans Krebs' work, Mitchell's contribution to cellular respiration was also recognized with a Nobel Prize in 1978.

You will use MitochondriaLab to simulate some of the pioneering experiments -- similar to those carried out by Szent-Gyorgi, Krebs, Mitchell and other researchers -- that were central to our understanding of cellular respiration. You will be adding substrates and inhibitors into a reaction flask containing isolated preparations of mitochondria and measure oxygen consumption as a function of the rate of these reactions. The challenge of MitochondriaLab will be to carefully analyze your results to develop an understanding of the sequence and control of these reactions.


  1. Campbell, M. K., Biochemistry, 2nd ed. NY: Saunders, 1995.

  2. Mathews, C. K., and van Holde, K. E. Biochemistry, 2nd ed. Menlo Park, California: Benjamin/Cummings, 1996.