Chapter 8 Microbial Metabolism

Understanding microbial metabolism is important for a wide variety of reasons, microbiologists can study bacterial metabolic pathways as a model for human pathways. Scientists can answer fundamental questions such as: How do cells gain energy to form cell structures? How do pathogens acquire energy and nutrients at the expense of the health of a patient? How does yeast turn grape juice into alcohol? Not only can we answer these questions by understanding microbial metabolism, we can also identify unknown microorganisms through biochemical testing. Biochemical tests allow microbiologists to identify end products from metabolic pathways, since not all bacterial species produce the same enzymes given the same substrate or “food” source the bacteria may or may not be able to utilize the substrate to grow. In this chapter you will learn how microbes use enzymes in metabolic pathways and how microbial cells metabolize glucose and produce energy for the cells needs.

Metabolism is the sum of all chemical reactions within a cell, these chemical reactions can either be catabolic (energy harvesting) or anabolic (biosynthetic) reactions. Catabolic reactions are processes used by cells to breakdown complex organic molecules into simpler compounds, during this process energy is released meaning the cell gains energy (Figure 8.1). An example of a catabolic reaction would be the required steps a cell must complete in order to breakdown glucose and gain Adenosine Triphosphate (ATP). Anabolic reactions within a cell use the ATP energy in order to build complex molecules from simpler molecules, an example of an anabolic reaction would be the steps a cell takes to build cell structures such as cell membranes or cell walls.

Figure 8.1 Metabolic action of a cell, showing the cyclical reaction between ATP and ADP. Nutrients are catabolized to make ATP while other cell processes use ATP. Image made by Author.

As referenced above in order for a cell to breakdown glucose and make ATP, there are a series of steps a cell must complete in order to do so. These so called “steps” are referred to as metabolic pathways or biochemical pathways (Figure 8.2).

EnzymesIn a metabolic pathway there are substrates, which are the starting compounds, intermediates, and end-products. Enzymes are involved in each step of a pathway which facilitate the formation of intermediates and end-products. Enzymes composed of protein and act as biological catalysts that lower the activation energy of a chemical reaction and therefore speed up the reaction (Figure 8.3). Some substrates are to large to be transported into the cell therefore certain cells can produce exoenzymes which are enzymes that are secreted from the cell and breakdown the substrate outside of the cell into simpler molecules which can then be transported inside the cell. Amylase is on such exoenzyme secreted by various bacteria, amylase hydolyzes starch (polysaccharide) into monosaccharides. Once the starch has been broken down outside of the cells the monosaccharides can then be utilized by the cell for growth. In the lab we can observe changes in media enriched with starch and determine which bacteria can produce the exoenzyme amylase.

Figure 8.2. Example of a linear biochemical pathway. Some pathways maybe branched or circular. Note were each arrow is located an enzyme would be involved in the formation of that product. Image made by Author.

Enzymes are substrate specific meaning a different substrate requires a different enzyme. Typically enzymes are named by adding “ase” to the end of the substrate, for example lipase would be a substrate specific enzyme that break down lipids. Enzyme specificity can be explained by a lock and key theory now commonly called the induced fit model. The key is the substrate and as you know the keys on your key chain are specific for only one lock. The lock is the enzyme and the substrate has a shape that fits into the enzymes active site. Once the substrate binds to the active site this is refered to the enzyme-substrate complex (Figure 8.4)

Figure 8.3. Example of how the use of an enzyme lowers the amount of energy for a reaction to take place. Note the time is also decreased for the same product to be made. Biochemical reactions within a cell could take place without enzymes however, life as we know it would not exist with out them.

Many enzymes are complete on their own however, some enzymes called apoenzymes have non-protein components called cofactors (Figure 8.5). Cofactors can be inorganic elements such as iron, magnesium, or zinc or they can be coenzymes which are derived from vitamins which can not be synthesized by certain organisms. For example, Escherichia coli can synthesize most of its own vitamins and convert them to coenzymes however, humans must consume vitamins inorder for proper cell metabolism. Since E. coli has a symbiotic relationship with humans vitamin K can be obtained from the E. coli

Figure 8.4. Schematic of enzyme action inside of a cell. Would this be a catabolic or anabolic reaction? Note the enzyme after the products are released, the enzyme is recycled in the cell and can bind to more substrate. (Kendall Hunt Image Figure 6.3 Energy and Metabolism)

living within our GI tract. The binding of the apoenzyme with its coenzymes and cofactors is refered to as a haloenzyme.

Some enzymes have sites separate from

the active site called an allosteric site. Depending on the enzyme certain molecules can bind to these sites which results in a change in the active site. There are some biochemical pathways in which the molecule that binds to the allosteric site would increase the performance of that enzyme in the pathway. However, we will focus on how enzyme activity can be inhibited from molecules binding to an allosteric site. When an enzyme is inhibited by a molecule binding to the allosteric site, molecule will actually change the shape of the active site (Figure 8.6a). When the active site shape is changed the substrate can no longer bind to the active site therefore, no products will be made. Cells can actually take advantage of these types of enzymes to regulate a metabolic pathway. A process called feedback inhibition allows a cell to shut down an entire biochemical pathway when the end product of the pathway acts as an allosteric inhibitor on the first enzyme in the pathway (Figure 8.6b). Escherichia coli can control the synthesis of isoleucine by this mechanism. The presence of isoleucine allosterically inhibits the first enzyme in the pathway, which will prevent the synthesis of isoleucine. Once isoleucine is depleted E. coli can resume production of the amino acid.

Figure 8.5 Anatomy of an apoenzyme. Image made by Author

Enzyme inhibitionEnzymes can be inhibited in a variety of ways as you will see, microbiologists can take advantage of understanding enzyme anatomy and function to develop antibiotics. If certain biochemcal pathways are shut down growth of the organism may cease. There are two main ways in which inhibitory molecules act on enzymes; competitively and noncompetitively. Competitive inhibiton refers to molecules that compete with the substrate for the active site. Once bound a competitive inhibtior prevents the substrate from binding and prevents the formation of end products (Figure 8.8). An example of a competitive inhibitor is the antibiotic sulfanilamide (commonly called sulfa), sulfanilamide is a competitive inhibitor that competes for the active site that normally binds with a molecule called PABA. PABA is converted into folic acid within the cell and is required for the synthesis of nucliec acids (DNA and RNA), if there is no folic acid then the cell cannot undergo cell replication. Sulfa drugs are selectively toxic since humans do not synthesize folic acid, we must absorb our folic acid from the foods we eat. Noncompetitive inhibitors attach to the allosteric site on enzymes there by altering the shape of the active site (Figure 8.9). There is not a specific example of an antibiotic that acts in this way however, heavy metals may bind to allosteric sites which explains some of the toxic effects metals have on not only bacteria but on humans as well.

Figure 8.6a. Image showing how allosteric inhibitors change the active site there by making the enzyme nonfunctional.Figure 8.6b. Feedback inhibition. Once the concentration of isoleucine is high enough inside the cell, isoleucine acts as an allosteric inhibitor on Enzyme 1 in the metabolic pathway to create isoleucine. The enzyme would be distorted as seen in 8.6a, thereby shutting the pathway down.(Kendall Hunt Image Figure 6.5 energy and metabolism)

Figure 8.8 This image shows how a molecule with a similar chemical structure or “shape” can bind to the active site of an enzyme. This is refered to as competitive inhibition since the inhibitior is competing with the substrate. Once the inhibitior is bound to the active site the reaction is stopped. Image made by Author

Figure 8.9 Noncompetitive inhibitiors bind to an allosteric site on the enzyme. Once a molecule is bound to the allosteric site the active site is distorted and the enzamatic pathway is shut down. Image made by Author.

Factors that influence enzymatic activityA cells ability to survive in extreme temperatures or pH is do to their enzymes ability to resist those conditions. A thermophile for example will have enzymes that are heat stable and therefore, allow the cell to grow in extreme temperatures. Enzyme activity can be influenced by environmental factors such as pH, temperature, salt, and substrate concentrations and have optimal activity ranges (Figure 8.10).

Figure 8.10. The effect of pH on enzyme activity.

How cells make ATPAs mentioned earlier ATP is the molecule cells use to perform cell processes. There is a cyclical role between ATP and ADP (adenosine diphosphate), ATP can be thought of as a charged battery and ADP as a “dead” battery. There are two processes used by heterotrophic bacteria to make ATP: substrate-level phosphorylation and oxidative phosphorylation. Phosphorylation refers to the addition of a phosphate to ADP (2 phosphates) to form ATP (3 phosphates). Substrate-level phosphorylation as the name suggests is when a cell uses a substrate or “food source” to phosphorylate ADP. Glycolysis and the Krebs cycle are examples of substrate-level phosphorylation and only a small amount of ATP is made. Oxidative phosphorylation harvests energy from the proton motive force, which will be discussed later, to add a phosphate to ADP.

Glucose metabolism: the basicsDuring a catabolic reaction one molecule (ex. Glucose) will act as an energy source or electron donor, when glucose is broken down by a cell to release energy glucose is oxidized. Oxidation refers to the loss of electrons, when glucose is oxidized another molecule must be reduced such as NAD+ or gain the electrons glucose lost in the process. What has just been described is a very basic oxidation reduction reaction (Figure 8.11).

Oxidation reduction reactions always occur simultaneously. With this example of glucose oxidation the electrons lost from glucose are transferred to electron carriers in the cell the two electron carriers involved in glucose metabolism during aerobic respiration in bacteria are NAD+ and FAD. These electron carriers will be reduced to fom NADH and FADH2 which will be refered to as reducing power (Table 1).

Table 1. The 2 most common electron carriers used by cells. When glucose is oxidized (loses electrons) the electron carriers are there to “grab” them, thereby becoming reduced.Electron Carrier Oxidixed Form Electron Carrier Reduced FormNAD+ NADHFAD FADH2

This reducing power is used to drive the electron transport system which in turn will create the proton motive force. Electrons from glucose are transferred to electron carriers and ultimately will combine with a terminal electron acceptor, in aerobic respiration this terminal electron acceptor is oxygen. When oxygen is used as the terminal electron acceptor the cell can produce the most ATP. During anaerobic respiration inorganic molecules other than oxygen, such as nitrate or sulfate, are used as terminal electron acceptors. Organisms that use an anaerobic process always yeild less ATP than if oxygen is used as the electron acceptor. Organisms that facultative anaerobes can either switch their metabolic process depending on what molecules are present in their environment or they are strictly fermenters. Certain genera of bacteria such as Streptococcus sp. are obligate fermenters and therefore do not respire. In the laboratory we will use thioglycolate media to help us determine an organisms oxygen requirements. However,

Figure 8.11. Oxidation-reduction reaction. Image made by Author.

we can only tell if the organism is a facultative anaerobe or an aerobe using this media therefore, in the laboratory activity following this chapter we will use oxidation/fermentation media (OF) to determine if the organisms ferment glucose or if they use the glucose through cell respiration.

Glucose metabolism: the processMicroorganisms oxidize sugars as their primary source of energy for anabolic reactions, glucose is the most common energy source. However, it should be noted that not all cells can use glucose as an energy source and rely on proteins or lipids for energy production. Energy can be obtained from glucose by respiration, which can be aerobic or anaerobic, or through fermentation, which is a process cells use that cannot respire. Considering glucose metabolism there are 3 metabolic pathways cells use to completely oxidize glucose, glycolysis, transition reaction or synthesis of acetyl CoA, and the Krebs Cycle. During these processes a small amount of ATP is made and reducing power is made which will be used to drive the proton motive force. We will now look at each of the pathways in more detail.

Aerobic oxidation of glucose chemical equation: C6H12O6 + 6 O2 6 CO2 + 6 H2O 38 ADP 38 ATP

GlycolysisAs observed in the chemical equation above glucose is a 6 carbon molecule, during glycolysis glucose is split into two 3 carbon molecules of pyruvate. In order to split glucose 2 ATP molecules are required during what is called the investment phase. The process of glycolysis is actually a 10 step process requiring many different types of enzymes, as a result the cell produces 4 ATP for a net gain of 2 ATP and has also created 2 NADH. Almost all cells that are capable of using glucose as an energy source use the process of glycolysis. Aerobic and anaerobic respiring bacteria use glycolysis as well as fermenters. However, cell respiration begins with the transition reaction since fermenters do not respire.

Cell Respiration: Transition ReactionThe transition reaction or synthesis of Acetyl-CoA reaction connects glycolysis to the Krebs cycle. From glycolysis we have two 3 carbon molecules of pyruvate which will feed into the transition reaction. During this reaction each of the pyruvate molecules will lose a carbon in the form of CO2. Since the pyruvate molecules are oxidized during the transition reaction 2 molecules of NAD+ will be reduced to NADH. At the end of the reaction there will be an end product of two 2 carbon Acetyl-CoA molecules.

Cell Respiration: Krebs CycleAcetyl-CoA from the transition reaction will start the Krebs cycle. As Acetyl-CoA is oxidized CO2 is given off as a byproduct for a total of 4 CO2. For each molecule of Acetyl-CoA the cell will gain 1 ATP, 3 NADH, 1 FADH2 for a total of 2 ATP, 6 NADH, and 2 FADH2. If you recall we started with a 6 carbon molecule of glucose lost 2 carbons in the form of CO2 during the transition reaction and lost 4 carbons during the Krebs

cycle as CO2, thus the carbon “backbone” of glucose is now completely gone yet we have only transformed a total of 4 ATP (2 from glycolysis and 2 from Krebs cycle). Next we will explore respiring cells make the majority of their ATP through oxidative phosphorylation.

Cell Respiration: Electron Transport System and the Proton Motive ForceAs glucose was oxidized you noticed that there was a fair amount of reducing power formed (NADH and FADH2). As NAD+ and FAD are reduced they carry the electrons to the cell membrane which is the site of the electron transport system (Figure 8.12). The electron carriers NADH and FADH2 will transfer the electrons, thereby becoming oxidized, to proteins in the cell membrane called cytochromes. There are numerous cytochromes invovled in the electron transport system, which will pass the electrons from one cytochrome to the next, as a result the electron energy is used to pump hydrogen ions or protons from the cell membrane. The oxidized electron carriers are shuttled back to glycolysis and the Krebs cycle to pick up more electrons therefore are recycled. The electrons eventually make their way back into the cell and combine with O2 and H+ to form H2O. As the positively charged hydrogen ions are pumped out of the cell they concentrate immediately outside of the cell membrane, there will be a net charge outside of the cell as positive therefore the inside of the cell has a net negative charge. The protons are “attracted” to the inside of the cell membrane however, the cell membrane is not permeable to the protons. This separation of charged ions creates an electrochemical gradient across the membrane. The electrochemical gradient represents potential energy refered to as the proton motive force. ATP is harvested when protons flow through a special turbine like protein called ATP synthase, ATP synthase phosphorylates ADP by oxidative phosphorylation. The theoretical yeild for ATP transformed from the proton motive force from the reducing power generated by glycolysis, transition reaction, and Krebs cycle using oxygen as a terminal electron acceptor is 34 ATP. Theoretically the cell gains 3 ATP from each NADH and 2 ATP from each FADH2. A total of 10 NADH and 2 FADH2 from the previous steps are used to drive the proton motive force.

FermentationThe process of fermentation occurs in organisms that cannot respire therefore they do not completely oxidize glucose using the transition reaction and Krebs cycle and do not have an electron transport system. Fermentation is a way cells can recycle NADH in the cell and create useful products for humans. Fermenters are indifferent toward oxygen meaning they do not use O2 to transform energy nor is O2 inhibitory toward the fermentation process. During fermenatitive metabolism organic molecules act as electron acceptors to recycle NADH. The fermentation process begins with glycolysis in which the cells gain (net) 2 molecules of ATP, 2 NADH, and will have two 3 carbon molecules of pyruvate at the end of the process. A popular fermentation process is alcohol fermentation in which alcohol is the end product of the process. Alcohol fermentation is performed by Saccharomyces cerevisiae which is a yeast (eukaryotic). Once the yeast split the 6 carbon molecule of glucose into pyruvate, pyruvate is oxidized to form acetylaldehyde (2 carbon) with CO2 as a by product. Acetylaldehyde will then gain electrons from the NADH produced from glycolysis to form alcohol and NAD+. As mentioned there are many useful products produced by fermentative metabolism, alcohol being one, organisms from the genus Clostridium can produce organic solvents such as acetone and isopropanol. Lactobacillus sp. produce lactic acid and is one of the organisms involved in making yogurt (Figure 8.13).

Figure 8.12 Electron Transport System occurring in the cell membrane of bacteria or inside the mitochondria in animal cells.