Exercise 2: Chymotrypsin—Active Site and Specificity
1. Obtain the amino acid sequences of bovine chymotrypsin and trypsin as follows. Go
to the National Center for Biotechnology Information website:
http://www.ncbi.nlm.nih.gov/. Select “Protein” from the dropdown menu and enter the
identification numbers for the two proteins (“gi” numbers) in the search box:
“576117, 60593450”. Click “Go.”
The entries for the two proteins should appear. From the “Display” dropdown menu,
select “FASTA.” You will then see the amino acid sequences of the proteins in FASTA
format. (This is a format in which the “>” symbol is followed by identification information
and a carriage return; the amino acid sequence, using the one-letter codes, begins after
that.)
Keep this window open so that you can copy the sequences during step 2.
Chymotrypsin and trypsin are both serine proteases. Name the three active site
residues of serine proteases that constitute the catalytic triad.
___________________________________________________________________
What chemical reaction do these enzymes catalyze?
___________________________________________________________________
Describe the differing specificities of chymotrypsin and trypsin for this reaction.
___________________________________________________________________
2. Open a new browser window and go to the EMBL-EBI Toolbox pairwise alignment
site: http://www.ebi.ac.uk/emboss/align/index.html. Copy and paste the sequences in
FASTA format into the two textboxes (include the “>” and identification information).
Leave all alignment parameters the same and click “Run.”
When your output appears, click the “Needle output” link to obtain a printer-friendly
version of the alignment. Print the alignment (you will turn in all alignments at the end).
Examine the sequence alignment, paying close attention to the residues of the catalytic
triad. You may wish to highlight these residues. Determine whether or not this is a
good alignment.
Click the “back” button on the browser window twice to go back to the pairwise
alignment site. Change the “Gap Open” and “Gap Extend” parameters and generate
another alignment. Try at least two different combinations and print the alignment each
time. Carefully compare all the alignments and determine which one is best and label it
“BEST” (you may decide that some alignments are roughly equal).
2
Use the space provided to answer the following discussion questions:
A. What constitutes a “good” alignment?
B. Why did you choose this particular alignment as the best relative to other alignments
you generated?
3. Chymotrypsin residues 189, 190, and 228 are three of the residues lining the
specificity pocket of the enzyme (where a side chain of the substrate binds). Use your
best sequence alignment to determine the identity of these residues for chymotrysin,
and then determine the identity of the corresponding residues for trypsin. (The residue
numbers will be different for trypsin.)
Chymotrypsin
Trypsin
189
____________
____________
190
____________
____________
228
____________
____________
Obtain the handout from the instructor which shows the structure of chymotrypsin.
3
Use the space provided to answer the following discussion question:
Although other features of these two enzymes are essential for their differing
specificities, the residues lining the specificity pocket make an important contribution to
their specificities. Residue 189 of chymotrypsin and the corresponding residue of
trypsin are each at the “base” of the specificity pocket, as you can see on the handout.
Reconcile the identity of this residue in chymotrypsin and trypsin with the differing
specificities of the two enzymes.
4. You have been working with bovine serine proteases so far. Choose an organism
that is distantly related to the cow (Bos Taurus) and search the NCBI protein database
for a chymotrypsin or trypsin sequence from this organism. (Go to
http://www.ncbi.nlm.nih.gov/ and choose “Protein” from the dropdown menu.) When
you find a sequence that looks interesting, click on the link to view the full entry. Read
the entry carefully to be sure that you have found a full-length sequence. (For instance,
50 amino acids is only a partial sequence.) Write down the gi number for the sequence
so that you will have it for step 5.
Use the space provided to propose some hypotheses before examining the
sequence further:
A. Predict whether or not the enzyme you chose will contain the residues forming the
catalytic triad. Explain your reasoning.
4
B. Predict the identities of the three residues lining the specificity pocket which you
examined in step 3 for bovine chymotrypsin and trypsin. Explain your reasoning.
5. You will now do a multiple sequence alignment for bovine chymotrypsin, trypsin, and
the serine protease you chose in step 4. You will need to obtain these three sequences
in FASTA format. If you already closed the browser windows where these sequences
were displayed, just go back to the NCBI website and use the gi numbers to find them.
Open a new browser window and go to the EMBL-EBI Toolbox ClustalW multiple
sequence alignment site: http://www.ebi.ac.uk/clustalw/index.html. Copy and paste the
three FASTA-format sequences into the textbox (paste the sequences one after the
other with a carriage return between them; include the > and identification information
for each sequence). Leave all alignment parameters the same and click “Run.”
When your output appears, click the “Alignment file” link to obtain a printer-friendly
version of the alignment. Print the alignment (you will turn in all alignments at the end).
Examine the alignment, paying careful attention to the residues of the catalytic triad and
the three residues lining the specificity pocket. If you are not satisfied with the
alignment, try it again changing the “Gap open,” “End gaps,” “Gap extension,” or “Gap
distances” parameters.
5
Use the space provided to answer the following discussion questions:
A. Were your hypotheses from step 4 correct? On your alignment, highlight the
residues of the catalytic triad and the three residues lining the specificity pocket. Name
any residues that turned out to be different from your predictions and discuss the
implications in terms of enzymatic activity and specificity.
B. Based on amino acid sequence alone, do you expect the serine protease from the
organism you chose to have chymotrypsin-like activity or trypsin-like activity? Justify
your answer.
6
C. Compare your multiple sequence alignment to that of two other groups who have
chosen a serine protease from a different organism; focus on the catalytic triad and the
three residues lining the specificity pocket. Were the identities of the three residues
lining the specificity pocket for the serine proteases chosen by the other groups different
from those of the serine protease you chose? Discuss any differences.
D. Looking at your multiple sequence alignment, are there any regions that are highly
conserved (many identical amino acids for all three sequences)? Are there any regions
that are not well conserved (few identical amino acids for all three sequences)? Do any
of these regions surround the residues of the catalytic triad or the three residues lining
the specificity pocket? How might the locations of any highly conserved regions relate
to the structure and function of the three enzymes?
When finished, turn in your sequence alignments and answers to the discussion
questions.