167Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
electrons! This means that the positively charged
nucleus of the hydrogen is no longer fully surrounded
by its negatively charged s-orbital and so, exhibits a
partial positive charge (ƍ+). Because the oxygen atom
now has a “little extra electron” from the hydrogens,
the oxygen has a slightly negative charge (ƍ-).
The partial positive charge of the hydrogen atoms can
now interact with the negative charges in other mole-
cules, just like you would observe with an ionic bond.
Because these are only “partial charges”, meaning
not “full plus” or “full minus” charge, they are not as
strong as ionic bonds.
It is very common for the hydrogen of one water mole-
cule to want to “hydrogen-bond” to the oxygen atom of
another water molecule. Hydrogen bonding is highly
prevalent in proteins and is the primary force that
causes proteins to fold into their three-dimensional
shape! Remember back to Chapter 1 when you learned
two DNA strands can come together to form the double
helix - it is hydrogen bonding that is important in the
complementary pairing of nucleotides (Figure 6-15).
It is hydrogen bonding between hydrogen-oxygen or
hydrogen-nitrogen that cause DNA to zip up into a
double helix. Further, in Chapter 4 when you learned
about transcription, it is hydrogen bonds between the
RNA transcript and the DNA (-) strand that hold RNA
polymerase locked-in to the DNA. Lastly, in Chapter
5 when you learned about translation, it is hydrogen
bonding that causes the tRNA’s anticodon to comple-
ment with the RNA transcript codon inside of the
ribosome, enabling translation to happen. Hydrogen
bonding is an essential kind of bond that allows The
Four B’s of Cell Operation to occur.
Van der Waals Interactions / London Dispersion
Force: The weakest of the intermolecular forces in
cells, these usually have bond strengths between 0.4
to 4 kJ/mol. As you learned earlier, the basic thermal
energy at room temperature is around 3 kJ/mol, and
so these interactions can be affected by the tempera-
ture of the environment.
Have you ever heated up glue in a glue gun, or steamed
a stamp to pull it off of the envelope? When doing this,
you are adding enough energy in the form of heat to
overcome the Van der Waals interactions and London
Dispersion Forces that are holding the glue molecules
together.
Just as hydrogen bonds were a weaker form of an ionic
bond because they involve partial charges, Van der
Waals interactions and London Dispersion Forces,
follow a similar principle and are even weaker. In
a hydrogen bond, the electron orbital around the
hydrogen atom is distorted by a nearby atom such
as another hydrogen, resulting in a positive charge
on one side of the hydrogen that can interact with
other (-) charged atoms (Figure 6-14). In the case of
Van der Waals interactions and London Dispersion
Forces, the electron orbitals in atoms aren’t perfectly
spherical and naturally uctuate. Also, sometimes it
just happens that there are more electrons on one
side of the atom than the other. When this happens,
one side of the electron orbital cloud is slightly more
negative in charge than the other side. Conversely, the
other side is slightly more positively charged. As you
probably already guessed, this means that the slightly
negative side of the atom can interact with a slightly
positive side of another atom (Figure 6-16).
This type of bond only happens when atoms come
into very close contact with one another, less than 0.6
picometers. However, if the atoms become too close
Figure 6-16. At very close proximities the uctuation the elec-
tron density of an atom can result in one side being more or
less charged than the other. Similar to an ionic bond or hy-
drogen bond, the slight negative charge of one atom can then
interact with the slight positive charge of another and cause
an interaction.
Unequal distribution
of electrons
Attractive force
Figure 6-15. Hydrogen bonding between C-G nucleotides.
Electronegative nitrogens cause hydrogen atoms to become
partially positive charged, enabling hydrogen bonding with
partially negatively charged nearby oxygens or nitrogens.
N
N
N
H
N
H
O
N
N
N
N
H
H
H
O
δ-
δ+ δ-
δ-δ+δ-
δ-δ+δ-
Book _genetic engineering hero-AUG2021.indb 167Book _genetic engineering hero-AUG2021.indb 167 8/18/21 12:03 PM8/18/21 12:03 PM
168 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
(less than 0.4 picometers), then repulsion between the
electron orbits occur and the atoms are repelled by the
negative charge of the electron of their orbitals.
When heat is added, the thermal energy causes the
electrons in the orbitals to be more lively. This makes
the electrons orbit more uniformally around the
nuclei of the atoms which breaks the +/- attractions.
Consider when you leave butter in the refrigerator. The
butter takes on a solid form because Van der Waals
interactions can occur at low temperature. If you leave
butter out in a hot room, the butter softens and even
melts. The room’s thermal energy causes the electron
orbitals in the butter molecule to become more lively,
which breaks the Van der Waals interactions allowing
the butter to ow freely. So next time you can’t butter
bread smoothly because the butter is too solid, you’ll
know who to blame!
Van der Waals interactions and London Dispersion
Forces are important in non-charged hydro-
phobic interactions like those that happen in the
phospholipid membrane in bacteria. During the
transformation in Chapters 4 and beyond, you’ll
recall that you rst kept your cells on cold and then
you increased the temperature to 42˚C to assist the
DNA in getting inside the cells, during what is called a
heatshock. When you increase the temperature, you
are increasing the thermal energy of the environment,
and this begins to overcome the Van der Waals inter-
actions and London Dispersion Forces that make the
lipids in the membrane stick together tightly and be
rigid. When heated, the uncharged tail groups of the
lipids slip and slide more freely, and the membrane
becomes more uid, allowing DNA plasmids to enter
more frequently.
If there is one key takeaway from this section, it is
that electrons of atoms drive the world around us. In
covalent bonds, it is the overlap of electron orbitals
that drives really strong bonds. In ionic bonds, it is
excess electron(s) or lack of electron(s) that makes
atoms positively or negatively charged and drives the
electromagnetic interaction. In hydrogen bonding, it
is the tugging of the hydrogens electron orbital that
causes it to be slightly positive and enables it to bind
to something with a negative charge. In Van der Waals
interactions it is the natural uctuation of electron
orbitals in atoms that can cause very weak positive/
negative charge interactions at very close range.
As you will now see, it is also the movement of elec-
trons that are at the heart of chemical reactions.
Protein enzyme catalysis in cells
Now that you have a much broader understanding
of what an atom is, what bonds are, and the basic
mechanism of enzymatic chemical reactions, we
can have a more in-depth look at one of the chemical
reactions that occurred when you transformed your
K12 E. coli cells with the DNA plasmid. Let’s look at
chloramphenicol acetyltransferase (CAT), the enzyme
that causes chloramphenicol resistance in the genetic
engineering experiments done throughout this book.
Figure 6-17. Chloramphenicol acetyltransferase bound to chloramphenicol (orange). Source: Protein Data Bank (PDB): 3cla; edited
using open source software, Chimera.
Book _genetic engineering hero-AUG2021.indb 168Book _genetic engineering hero-AUG2021.indb 168 8/18/21 12:03 PM8/18/21 12:03 PM
169Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
When the chloramphenicol molecule is processed by
CAT, it is no longer active and no longer able to harm
the cells.
In Figure 6-17 the CAT protein (beige) is interacting
with chloramphenicol (orange). Chloramphenicol is in
the “binding pocket” or active site of CAT and held in
place with hydrogen bonds formed between the amino
acid side chains in the protein, water molecules, and
chloramphenicol (Figure 6-18). Each of the thin blue
lines indicates a hydrogen bond. If you’ve forgotten
what amino acids look like, check back to Figure 3-30.
In Figure 6-18, the amino acid side chains of CAT
(pink) are also able to bind to a second molecule called
acetyl-CoA (not shown). Remember acetyl-CoA? This
molecule is also an essential part of the Atf1 reaction
for making overripe banana smell!
The chemical reaction that CAT catalyzes involves
the amino acid histidine (Figure 6-18 (pink)), which is
an important part of initiating the chemical reaction
(Figure 6-19):
(a) A nitrogen atom in a histidine amino acid (pink)
uses some “spare electrons” to “steal” a hydrogen
from the chloramphenicol. This is shown by an
arrow from two red dots (electrons) “reaching out
and stealing” the hydrogen from the chloramphen-
icol molecule (green);
(b) The electrons that previously formed the bond
with the now “stolen” hydrogen, form a new bond
with the carbon atom of the acetyl-CoA (blue),
which is also bound in the enzyme binding pocket.
This is shown by the arrow continuing to the
carbon in acetyl-CoA;
(c) Step (b) causes the bond from the double
carbon-oxygen in acetyl-CoA to be broken, and the
electrons move to the oxygen;
(d) The electrons only stay temporarily on the
oxygen and quickly move back to reform the
carbon-oxygen double bond;
(e) The movement of electrons in (c) and (d) cause
the carbon-sulfur bond electrons to move to the
sulfur atom of the CoA molecule, ‘destroying’ the
carbon-sulfur bond;
(f) The acetate group is free and separate from the
“CoA”. The carbon that was previously bound to
the sulfur of the CoA forms a stable covalent bond
with the available oxygen on the chloramphenicol
molecule.
Several factors contribute to the CAT/chlorampheni-
col chemical reaction happening. In one instance, the
substrates chloramphenicol and acetyl-CoA are put
into very close proximity with one another by bump-
ing around the cell and then binding simultaneously
to CAT. The hydrogen bonding formed between the
substrates and CAT hold the substrates in place, but
they also have an influence on the bonds (electron
orbital clouds) in the substrates which changes elec-
tronic structure of the substrates. The histidine amino
acid of CAT is ideally positioned to stir up trouble and
start the chemical reaction by using its extra electrons
to steal the hydrogen from chloramphenicol, which
starts a chain reaction of ‘electron movement’ events.
These are themes that occur in protein enzymes.
In many chemistry and biochemistry textbooks, you
will learn about how enzymes “lower the activation
energy” required for a chemical reaction to occur.
This is another way of saying that to break a covalent
bond which has 300 kJ/mol energy you typically need
300 kJ/mol of energy. However, enzymes are able to
lower the energy required to break the bond. Enzymes
“change the rules” by employing some of the mecha-
nisms you just learned about. If you haven’t already,
go back to the Chemicals Reaction Going Deeper 3-7
which makes the analogy between a roller coaster and
a chemical reaction. In this instance, the ability of CAT
to initiate a chain of electron jumping events is the
“tunnel” in the Going Deeper analogy (Figure 3-29).
These electron jumps cause the creation and breaking
of bonds without the addition of 300 kJ/mol energy.
Figure 6-18. Zoom in of the chloramphenicol binding pock-
et of CAT and chloramphenicol. The blue lines are hydrogen
bonds. The hydrogen bonds holding chloramphenicol in the
binding pocket are between the amino acid side chains, water
molecules (red spheres), and the chloramphenicol molecule.
The histidine amino acid in the chemical reaction is in pink.
Book _genetic engineering hero-AUG2021.indb 169Book _genetic engineering hero-AUG2021.indb 169 8/18/21 12:03 PM8/18/21 12:03 PM
170 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
X-ray Crystallography Going Deeper 6-7
You might be wondering where the protein structure illustrations in Figure 6-17 and Figure 6-18 come from
and why acetyl-CoA is not in Figure 6-17. The illustrations in these gures are based on real data! But how
can scientists possibly take pictures of proteins at the atomic scale? How can you zoom in that far?
Scientists use a technique where they rst create proteins through genetic engineering, much in the way that
you did with the various proteins you engineered your K12 E. coli to produce. They then purify the proteins,
and treat them to different chemical conditions and dry the samples out so that the proteins form crystals.
Crystals are materials that are made up of repeating patterns. Scientists then shoot X-rays through the crys-
tals and look at how the X-rays are scattered and diffracted by the proteins in the crystals. The diffraction
patterns tell scientists where the different atoms of the proteins are, and using software like Chimera, you
can visualize the atoms and the molecules (https://www.cgl.ucsf.edu/chimera/). This technique is called X-ray
Crystallography, and it is what Rosalind Franklin used to get one of the best X-ray images of DNA, which led
to the discovery of its structure in 1953.
There is no acetyl-CoA in the illustrations because no scientist has been able to successfully create crys-
tals that have CAT, chloramphenicol, and acetyl-CoA. While you might nd it easy to create water crystals
by simply putting water in the freezer, it can take years for scientists to formulate the right experimental
conditions to cause the proteins and the substrates to form crystals!
Atf1 Video Breakout
Now that you completed the Enzymatic Processing hands-on exercise and have learned about enzyme
catalysis, have another look at the video covering the chemical reaction to create banana smell. You’ll have
a deeper appreciation for the breaking and making of covalent bonds by Atf1. Visit: https://amino.bio/atf1
Figure 6-19. The CAT/chloramphenicol chemical reaction includes several steps that involve the movement of electrons. The arrows
in this diagram indicate where electrons, and therefore bonds, move. Chloramphenicol (green); histidine amino acid of CAT (pink);
acetyl-CoA (blue). When the acetyl (blue) is connected to chloremphenicol (green), it no longer has antibiotic properties.
C
H
O
2
N
OH
O
H
C
N
H
C
CHCl
2
O
CH
2
O
H
N
N
CH
3
C
S
CoA
H
(a)
(e)
(f)
(b)
(c)
(d)
Chloramphenicol
His195
Book _genetic engineering hero-AUG2021.indb 170Book _genetic engineering hero-AUG2021.indb 170 8/18/21 12:03 PM8/18/21 12:03 PM
171Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Enzyme processing in cells:
Amino acids
You’ve been learning about enzyme processing in the
context of enzymes that you’ve engineered the cells
to produce. However, as has been mentioned, cells
already rely heavily on protein enzymes for normal
operations. Let’s take a quick look at proteins and the
amino acids that they are made up of in the context of
enzymatic processing. Where do amino acids come
from? From enzymatic processing!
Using the Three Steps to Microfacturing, E. coli cells
complete a series of enzymatic reactions to modify
sugars and other small chemical molecules into
amino acids. Another name for this is biosynthesis.
In the exercises in this chapter you completed simple
chemical reactions (Figure 6-20, top) where one or two
substrates were converted into products by a single
enzyme. However, cells often do complex chemical
reactions that require more than one enzyme to cause
a chain of chemical reactions where the product(s)
of one enzyme reaction becomes the substrate(s) of
another (Figure 6-20, bottom). E. coli cells use complex
enzymatic reactions to create amino acids.
In Figure 6-21 you will see a real amino acid biosyn-
thesis map which shows many different chemical
reactions. Within E. coli cells, all chemical reactions
in the map happen simultaneously and result in the
creation of amino acids. At rst glance, it looks very
complicated but, when you understand how it works,
it’s quite simple. As simple as using Google Maps to get
directions from Point A to Point B:
• The open circles are a molecule, like a sugar, a fat,
or amino acid (not proteins).
•
The arrows represent a chemical reaction and its
direction. The molecule at the base of the arrow is
converted into the molecule at the tip of the arrow.
•
In general, the arrow also represents a protein
enzyme that causes that chemical reaction to
happen.
Find a molecule in Figure 6-21 called Fructose-6P
(Fructose-6-Phosphate) on the top left. Where have
you heard of fructose before? You’ve probably heard
of high fructose corn syrup, the sugar that comes from
corn and is used to sweeten food in North America.
Corn syrup is one form of fructose that can be found
in candy, soda pop, Slurpees, and a lot more. Bacte-
ria also enjoy sweet things. In fact, they use sugar
from their environment, some of it being fructose,
to make amino acids. For example, the E. coli in your
large intestine will directly use some of the fructose
from your candy and turn it into amino acids your
body needs - perhaps you can use that as an excuse to
eat more candy! In the case of the Figure 6-21, Fruc-
tose-6-Phosphate is a fructose sugar molecule that
was taken into a cell and slightly modied by enzymes
called fructokinase or hexokinase to have a phosphate
added to carbon C6 of the sugar molecule. Fructose-6P
can be converted to many other molecules by many
different enzymes, including being converted into
amino acids.
Using Figure 3-30, you can correlate the structure of
the amino acids within the biosynthesis pathway in
Figure 6-21. You can see that it takes many chemical
reactions to convert fructose-6P to an amino acid.
Figure 6-20. Cells process many substrates into “products” through microfacturing, more formally known as biosynthesis. In simple
reactions, a substrate is turned into a product with a single enzyme (E). In complex pathways, several enzymes (E) are involved, and
the products of one enzyme reaction become the substrates of another enzyme reaction.
Simple Reaction
Enzyme 1
Substrate
(starting material
/ raw material)
Substrate Intermediate
Product 1
Product
(end material)
Complex Reaction
E1
Intermediate
Product 2
E2
Intermediate
Product 3
E3
End Product
E4
Book _genetic engineering hero-AUG2021.indb 171Book _genetic engineering hero-AUG2021.indb 171 8/18/21 12:03 PM8/18/21 12:03 PM
..................Content has been hidden....................
You can't read the all page of ebook, please click here login for view all page.