Download Bis2A 5.5: Fermentation and regeneration of NAD+

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Bis2A 5.5: Fermentation and
regeneration of NAD+
∗
The BIS2A Team
This work is produced by OpenStax-CNX and licensed under the
Creative Commons Attribution License 4.0†
Abstract
This module will discuss the process of fermentation and the purpose of fermentation.
Section Summary
This section discusses the process of fermentation. Due to the heavy emphasis in this course on central carbon
metabolism the discussion of fermentation understandably focuses on the fermentation of pyruvate. Nevertheless, some of the core the principles that we cover in this section apply equally well to the fermentation
of many other small molecules.
The Purpose of Fermentation
The oxidation of a variety of small organic compounds is a process that is utilized by many organisms to garner energy for cellular maintenance and growth. The oxidation of glucose via glycolysis is one such pathway.
Several key steps in the oxidation of glucose to pyruvate involve the reduction of the electron/energy shuttle
NAD+ to NADH. At the end of section 5.3 you were posed with the challenge of trying to gure out what
options the cell might reasonably have to re-oxidize the NADH to NAD+ in order to avoid consuming the
available pools of NAD+ and thus stopping glycolysis. Put dierently, during glycolysis cells can generate
large amounts of NADH and slowly exhaust their supplies of NAD+ . If glycolysis is to continue, the cell
must nd a way to regenerate NAD+ , either by synthesis or by some form of recycling.
In the absence of any other process - that is, if we consider glycolysis alone - it is not immediately obvious what the cell might do. One choice is to try putting the electrons that were once stripped o of the
glucose derivatives right back onto the downstream product, pyruvate or one of its derivatives. We can
generalize the process by describing it as the returning of electrons to the molecule that they were once
removed from, usually to restore pools of a oxidizing agent. This, in short, is fermentation. As we will
discuss in a dierent section, the process of respiration can also regenerate the pools of NAD+ from NADH.
Cells lacking respiratory chains or in conditions where using the respiratory chain is unfavorable may choose
fermentation as an alternative mechanism for garnering energy from small molecules.
Helpful Videos
Here is a chemwiki link on fermentation reactions1 .
∗ Version
1.1: Jan 21, 2016 11:55 pm -0600
† http://creativecommons.org/licenses/by/4.0/
1 http://cnx.org/content/m59694/latest/ http://chemwiki.ucdavis.edu/Biological_Chemistry/Metabolism/Glycolysis/Fermentation
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1 An Example: Lactic Acid Fermentation
An everyday example of a fermentation reaction is the reduction of pyruvate to lactate by the lactic acid
fermentation reaction. This reaction should be familiar to you, it occurs in our muscles when we exert
ourselves during exercise. When we exert ourselves our muscles require large amounts of ATP to perform
the work we are demanding of them. As the ATP is consumed, the muscle cells are unable to keep up with
the demand for respiration, O2 becomes limiting and NADH accumulates. Cells need to get rid of the excess
and regenerate NAD+ , so pyruvate serves as an electron acceptor, generating lactate and reducing NADH
to NAD+ . Many bacteria use this pathway as a way to complete the NADH/NAD+ cycle. You may be
familiar with this process from in products like sauerkraut and yogurt. The chemical reaction of lactic acid
fermentation is the following:
Pyruvic acid + NADH ↔ lactic acid + NAD+
note:
Figure 1: Lactic acid fermentation converts pyruvate (a slightly oxidized carbon compound) to lactic
acid. In the process, NADH is oxidized to form NAD+ .
Source:
modications of http://polymerinnovationblog.com/from-corn-to-poly-lactic-acid-plafermentation-in-action/
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Energy Story for Fermentation of Pyruvate to Lactate
An example (if a bit lengthy) energy story for lactic acid fermentation:
The reactants are pyruvate, NADH and a proton. The products are lactate and NAD+ . The process of
fermentation results in the reduction of pyruvate to form lactic acid and the oxidation of NADH to form
NAD+ . Electrons from NADH and a proton are used to reduce pyruvate into lactate. If we examine a table
of standard reduction potential we see under standard conditions that a transfer of electrons from NADH to
pyruvate to form lactate is exergonic and thus thermodynamically spontaneous. The reduction and oxidation
steps of the reaction are coupled and catalyzed by the enzyme lactate dehydrogenase.
2 A second example: Alcohol Fermentation
Another familiar fermentation process is alcohol fermentation, which produces ethanol, an alcohol. The
alcohol fermentation reaction is the following:
Figure 2: Ethanol fermentation is a two step process. Pyruvate (pyruvic acid) is rst converted into
carbon dioxide and acetaldehyde. The second step, converts acetaldehyde to ethanol and oxidizes NADH
to NAD+ .
In the rst reaction, a carboxyl group is removed from pyruvic acid, releasing carbon dioxide as a gas
(some of you may be familiar with this as a key component of various beverages). The second reaction
removes electrons from NADH, forming NAD+ and producing ethanol (another familiar compound - usually
in the same beverage) from the acetaldehyde, which accepts the electrons.
Write a complete energy story for alcohol fermentation. Propose possible benets of this
type of fermentation for the single celled yeast organism.
note:
3 Fermentation pathways are numerous
While the lactic acid fermentation and alcohol fermentation pathways described above are examples, there
are many more reactions (too numerous to go over) that Nature has evolved to complete the NADH/NAD+
cycle. It is important that you understand the general concepts behind these reactions. In general, cells try
to maintain a balance or constant ratio between NADH and NAD+ ; when this ratio becomes unbalanced,
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the cell compensates by modulating other reactions to compensate. The only requirement for a fermentation
reaction is that it uses a small organic compound as an electron acceptor for NADH and regenerates NAD+ .
Other familiar fermentation reactions include, ethanol fermentation (as in beer and bread) and propionic
fermentation (it's what makes the holes in swiss cheese) and malolactic fermentation (it's what gives chardonnay is more mellow avor, more conversion of malate to lactate the softer the wine). In Figure 3 below
you can see a large variety of fermentation reactions that various bacteria use to reoxidize NADH to NAD+ .
All of these reactions start with pyruvate or a derivative of pyruvate matabolism, such as oxaloacetate, or
formate. Pyruvate is produced from the oxidation of sugars (glucose or ribose) or other small reduced organic
molecules. It should also be noted that other compounds can be used as fermentation substrates besides
pyruvate and its derivatives. These include: methane fermentation, sulde fermentation, or the fermentation
of nitrogenous compounds such as amino acids. You are not expected to memorize all of these pathways.
You are, however, expected to recognize a pathway that returns electrons to products of the compounds that
were originally oxidized to recycle the NAD+ /NADH pool and to associate that process with fermentation.
Figure 3: Various fermentation pathways using pyruvate as the initial substrate. In the gure, glucose is oxidized to pyruvate and pyruvate is the starting material (substrate) for a variety of dierent
fermentation reactions.
A note on the link between substrate level phosphorylation and fermentation
Fermentation occurs in the absence of molecular oxygen (O2 ). It is an anaerobic process. Notice there is no
O2 in any of the fermentation reactions shown above. Many of these reactions are quite ancient, hypothesized
to be some of the rst energy generating metabolic reactions to evolve. This makes sense if we consider the
following:
1. The early atmosphere was highly reduced, with little molecular oxygen readily available.
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2. Small, highly reduced organic molecules were relatively available, arising from a variety of chemical
reactions.
3. These types of reactions, pathways and enzymes are found in many dierent types of organisms,
including bacteria, archaea and eukaryotes, suggesting these are very ancient reactions.
4. The process evolved long before O2 was found in the environment.
5. The substrates, highly reduced small organic molecules, like glucose, were readily available.
6. The end products of many fermentation reactions are small organic acids, produced by the oxidation
of the initial substrate.
7. The process is coupled to substrate level phosphorylation reactions. That is, small reduced organic
molecules are oxidized and ATP is generated by rst a red/ox reaction followed by the substrate level
phosphorylation.
8. This suggests that substrate level phosphorylation and fermentation reactions co-evolved.
If the hypothesis is correct, that substrate level phosphorylation and fermentation reactions
co-evolved and were the rst forms of energy metabolism that cells used to generate ATP, then
what would be the consequences of such reactions over time? What if these were the only forms of
energy metabolism available over hundreds of thousands of years? What if cells were isolated in a
small closed environment? What if the small reduced substrates were not being produced at the
same rate of consumption during this time?
note:
Consequences of fermentation
Imagine the world where fermentation is the primary mode for extracting energy from small molecules. As
populations thrive, they reproduce and consume the abundance of small reduced organic molecules in the
environment, producing acids. One consequence is the acidication (decrease of pH) of the environment,
including the internal cellular environment. This is not so good, since changes in pH can have a profound
inuence on the function and interactions among various biomolecules. Therefore mechanisms needed to
evolved that could remove the various acids. Fortunately, in an environment rich in reduced compounds,
substrate level phosphorylation and fermentation can produce large quantities of ATP.
It is hypothesized that this scenario was the beginning of the evolution of the F0 F1 ATPase, a molecular
machine that hydrolyzes ATP and translocates protons across the membrane (we'll see this again in the
next section). With the F0 F1 ATPase, the ATP produced from fermentation could now allow for the cell to
maintain pH homeostasis by coupling the free energy of hydrolysis of ATP to the transport of protons out
of the cell. The down side is that now cells are pumping all of these protons into the environment, which
will now start to acidify.
If the hyposthesis is correct, that the F0 F1 ATPase also co-evolved with substrate level
phosphorylation and fermentation reactions, then what would happen over time to the environment?
While small reduced organic compounds may have been initially abundant, if fermentation "took
o" at some point the reduced compounds would run out and ATP might then become scarce as
well. That's a problem. Thinking with the design challenge rubric dene the problem(s) facing the
cell in this hypothesized environment. What are other potential mechanism or ways Nature could
overcome the problem(s)?
note:
Exercise 1
What reactants are used up in glycolysis (need to be replaced)?
a.
b.
c.
d.
e.
NAD+
NADH
ATP
ADP
glucose
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(Solution on p. 7.)
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g.
h.
i.
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glycolytic enzymes
a, c and d
a and e
all of the above
Exercise 2
(Solution on p. 7.)
Which reactants that are used up in glycolysis are "replaced" by the act of fermenting?
a.
b.
c.
d.
e.
f.
g.
h.
i.
NAD+
NADH
ATP
ADP
glucose
glycolytic enzymes
a, c and d
a and e
all of the above
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Solutions to Exercises in this Module
Solution to Exercise (p. 5)
h
Solution to Exercise (p. 6)
a
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