13 Jan environmental parameters and

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Introduction to Cell and Molecular Biology
Biology 211 Lab 6 and 7
Enzymes and Enzymatic Reactions
All living things interact with the environment, use energy, and give off waste.
To carry out these and other life processes, biochemicals must be made,
transformed and eventually broken down.
These biochemical reactions are rapid, specific and occur at temperatures
compatible with life. What makes these reactions possible are a class of globular
proteins called enzymes. Without these enzymes, life processes are not possible.
Learning Objectives:
Upon completion of this lab you should be able to:
1. Define:
a. Enzyme
b. Catalyst
c. Substrate
d. Product
e. Active site
f. Activation energy
g. Blank
h. Control
i. Reaction velocity
j. Reagent
k. Slope
l. Competitive inhibitor
m. Non-competitive
inhibitor
2. Describe the physical characteristics of an enzyme.
3. What environmental factors affect enzyme activity and how do they exert their
effect?
4. Use your pipetting and measuring skills to set up an experiment to test
enzyme activity.
5. Collect absorbance data using a spectrophotometer.
6. Construct a line (“connect the dots”) graph of the scientific data using
Excel™.
7. Interpret a graph of the data and compare to other graphs.
8. Understand why is a blank important in using the Spectrophotometer.
9. Understand why is a control important in experiments determining enzyme
activity.
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Introduction to Enzymes:
Enzymes have several important characteristics:
? Enzymes are able to take a specific biomolecule called substrate (or substrates
because some enzymes use more than one substrate) and change it into
another specific biomolecule called product (or products).
? Enzymes have what is called an active site. An active site is a pocket or cleft in
the protein that is shaped so that it specifically fits a certain substrate. It is here
that the substrate is converted into a product.
? Enzymes are biological catalysts. They participate in a biological reaction and
guide the reaction, but they are not changed by the reaction. Since they are not
changed by the reaction they are used over and over to carry out the same
reaction.
? Enzymes speed up reaction rate by decreasing the activation energy required
to start the reaction. Activation energy is the energy required to break certain
bonds in the substrate so that other bonds can form. The formation of these new
bonds results in the formation of the product.
? Enzymes, like all proteins, are affected by the environment. Changes in
temperature, pH and the ionic strength of the surrounding solution all affect
enzyme activity. For example, there are some bacteria that have adapted to life
on coal and iron ore “mine tailings”. Tailings are the materials left over after the
coal or iron ore has been removed. When mixed with water, these tailings
commonly have a pH of 2.5 or less and yet Thiobacillus bacteria live quite
happily on it.* What do you think the optimal pH for activity of the enzymes of
these bacteria will be? If you said about pH 2.5 you have the idea! So what do
you think would happen if you tried to grow these “acidophilic” bacteria at a
neutral pH? The answer? Certain death, because the enzymes of acidophilic
bacteria are not adapted a pH of 7. However, most organisms have enzymes
that function best between pH 5 and pH 9.
*Run off from these tailings is a serious source of pollution in mining districts of the
Eastern US. You may be interested to know that there are also bacteria that thrive at
excessive temperatures and highly salty conditions by having enzymes that have
evolved in these extreme environments.
? Certain chemicals, as well as environmental factors can also inhibit enzyme
activity. Some chemicals inhibit enzymes by attaching reversibly to sites on the
enzyme other than the active site (allosteric sites) resulting in a conformational
change to the enzyme so that the enzyme is unable to function. These inhibitors
are known as non-competitive inhibitors because they are able to inhibit an
enzyme without competing with the normal substrate for the active site.
? Other chemicals are so similar to the enzyme substrate that they can bind to the
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active site of the enzyme. Since the chemical is not the normal substrate, it can
not be converted into the product. These particular chemicals do not
permanently bind to the enzyme and therefore they inhibit the enzyme by
competing with the normal substrate for the active site. You can probably guess
that these inhibitors are called competitive inhibitors.
So what enzyme are you going to use in today’s experiment? Since all living
things use enzymes, there are a wide variety of enzymes in nature. For example, there
are 100,000 or more available from the human body alone. But the enzyme we will use
is called alkaline phosphatase. It is found in many different organisms and it catalyses
the removal of a phosphate group from DNA molecules. The reaction works something
like this:
Alkaline Phosphatase
DNA with phosphate attached DNA without phosphate attached + phosphate
(substrate) (products)
You will not use real DNA as a substrate in this experiment because it is difficult to
measure the removal of the phosphate group. We will use another compound called
paranitrophenyl phosphate as the substrate. The reaction looks like this:
Alkaline Phosphatase
Paranitrophenyl phosphate Nitrophenyl + phosphate
(colorless substrate) (yellow product)
The reason we use this reaction is that the removal of the phosphate group from
the substrate (paranitrophenyl phosphate) can be easily measured because the
paranitrophenyl phosphate is colorless and one of the products, nitrophenol, is a bright
yellow color. For this reason it is easy to follow the progress of this reaction with the
spectrophotometer. The Spec 20 is used to measure the change in absorbance as the
reaction proceeds. In this case, as more nitrophenol is produced the solution becomes
a darker yellow and the absorbance increases.
One note about nitrophenol. It is a possibly hazardous material and contact with
skin should be avoided. Wash thoroughly if contact does occur.
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The Experiments:
Enough background, let’s talk about the experiment we are going to do today!
Part I: You are going to set up reactions in test cuvettes which can measure the
formation of product by the enzyme. You will use 5 different concentrations of the
enzyme to determine the optimal concentration of the enzyme to use in further
experiments. You will correctly set up the reagents, run the experiment and graph the
results. Then you will compare the results of the 5 different samples and choose the
optimal enzyme concentration.
Part II: In Part I you found out how much enzyme to use and have seen how well it
works under “normal” conditions. Next you are going to change some of the
environmental parameters and see how that affects enzyme activity.
Experimental Procedures
(all experiments are run at room temperature except where noted)
Part I: Find the optimal concentration of enzyme
Read all of Part I. You should do this before starting any experiment. It allows
you to anticipate what is coming up and lets you avoid nasty surprises.
Blanks and controls are quite important in experiments. The reason for having
blanks and controls are several. Firstly, when using the spectrophotometer you are
trying to measure the formation of product in the experimental cuvettes. Unfortunately
there are several other reagents in the cuvettes that are necessary for the reaction to
occur (like the buffer, the substrate, and the enzyme itself) that are not the chemical
that we are trying to measure. We are only interested in changes in the absorbance
due to the substrate being changed into product. Therefore, if we can exclude these
other chemicals from our spectrophotometer reading we should get clearer results. The
blank is a way of doing exactly that. The blanks in this experiment include all of the
reagents except the enzyme.
The blank often serves another purpose in experiments like this one. Sometimes
there may be a color change in the experimental tubes due to oxidation of the