Download Chapter 8 Microbial Metabolism

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).
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
In 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 8.3. 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.
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 enzymesubstrate complex (Figure 8.4)
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)
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
living within our GI tract. The binding of the apoenzyme with its coenzymes and
cofactors is refered to as a haloenzyme.
Figure 8.5 Anatomy of an apoenzyme. Image made by Author
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 8.6a. 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)
Enzyme inhibition
Enzymes 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 8.8 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 activity
A 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 ATP
As 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 basics
During 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).
Figure 8.11. Oxidation-reduction reaction. Image made by Author.
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 Form
NAD+
NADH
FAD
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,
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 process
Microorganisms 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
Glycolysis
As 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 Reaction
The 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 Cycle
Acetyl-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 Force
As 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.
Figure 8.12 Electron Transport System occurring in the cell membrane of
bacteria or inside the mitochondria in animal cells.
Fermentation
The 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).