Transcript PowerPoint Presentation - Berkeley Cosmology Group

Soybean Lipoxygenase:
Which amino acids matter?
By: Noreen Castellanos, Robert
Zuniga, Gerardo Gallardo
Soybean Lipoxygenase
Enzyme
- A kind of protein that help a reaction go faster.
Amino acids
- Are the building blocks of proteins.
- There are 20 different kinds of amino acids.
Hypothesis
In this experiment, we tested
which amino acids are
important to the rate of
the enzyme soybean
lipoxygenase.
- We tested a wild type
and two mutants.
- One enzyme (I553W) was mutated near the active site.
The active site is where the reaction occurs. The other
mutant (L496F) was mutated far away from the active site.
- We hypothesize that the amino acid closer to the
active site is more important than the one further away.
Experiment
- To test our hypothesis we will purify each enzyme.
- We will measure the rate of reaction of each enzyme.
- If the reaction rate is slower for a
mutant, then it shows that there is
a major effect if we change that
particular amino acid.
- So if our hypothesis is correct
then the reaction rate of the
mutant closer to the active site
(I553W) would be slower
compared to the reaction rate of
the wild type and the other
mutant.
Centrifuge and Lysis
In our experiment, we wanted
purified enzymes.
- To obtain purified enzymes we
had to break open bacterial cells
which is known as lysis.
- To remove all the debris (such as
cell walls), we centrifuged our
cells. All the debris went to the
bottom and all of the proteins
stayed on the top.
Affinity Chromatography
At this point we still need to get
our enzyme by itself.
- We poured the mixture of protein
into a column
- The tag of our enzyme sticks to the
beads in the column.
- Then we poured buffer to clean out
all the impurities. (Wash Step)
- Finally, we poured imidazole that
breaks the bond between our
enzyme and the beads, which
gives us our enzyme by itself.
(Elution Step)
Gel Electrophoresis
Gel electrophoresis is a technique we used to
determine if our purification worked.
- We load our samples on the top.
- Light proteins will move down quickly, while giant
proteins will not be as fast.
Data: what does our gel tell us?
Our gel tells us that our purification worked!
In the wash lanes, we see all of our impurities.
In the elution lanes, we see a single band that represents
our pure enzyme! (soybean lipoxygenase)
Wild Type
L496F
I553W
elution
wash
How do we measure a reaction rate?
- Linoleic Acid Hydroperoxide is the product that
absorbs the light and what allows us to see the rate of
the reaction.
Linoleic Acid
Hydroperoxide
- Because the product absorbs light at 234nm, we could
use a spectrophotometer to measure the rate of
reaction for each enzyme.
- A spectrophotometer is a machine that measures the
absorbance properties of a sample.
Data: What rates did we actually see?
- The wild-type reaction went the
fastest, enzyme L496F was 5 times
slower, and enzyme I553W was really
slow (50x!).
- The main reason that enzyme
I553W was the slowest is because
the mutation was closest to the active
site and therefore it was more
important for the reaction.
-The mutation in enzyme L496F was
farther from the active site and it was
not as necessary to carry out the
chemical reaction.
Wild-type Enzyme
L496F Enzyme
I553W Enzyme
Summary: Was our hypothesis correct?
Original Hypothesis: The closer a mutation was
to the active site the more necessary it was for the
reaction.
Conclusion: Our
hypothesis was proven
right by our experiment
because we saw that the
enzyme with the mutation
closest to the active site
was the slowest.