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PathwayTools Workshop:
Exercise 2: Assigning enzymes and creating protein complexes
In this exercise you will:
1. Assign enzymatic activities to proteins
2. Define protein complexes
3. Create a publication frame
4. Export a pathway to a file
Disclaimer: In this exercise we will create enzyme monomers and
complexes that are completely fictitious. The enzymatic activities
described are not based on real data.
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Please only modify the H. pylori database.
Save your work.
Open the L-ascorbate degradation pathway (created in the last exercise) in
the Navigator.
1. Assigning enzymatic activity to a protein. Right click on the first reaction in
the pathway, and choose “Edit -> Create/Add Enzyme”. Click the “Search by
Genes or Create New Protein” button. Specify “polypeptide” and type in
“HP1580” for the gene name. Click OK.
The protein Editor opens up. Scroll down and locate the space for defining the
“Enzyme activity name”. Type “ascorbate hydratase”. Familiarize yourself with
the different fields of this editor.
2. Defining a protein complex and assigning activity to it. Right click on the
second reaction in the pathway, and choose “Edit -> Create/Add Enzyme”. Click
the “Search by Genes or Create New Protein” button. Specify “protein complex”.
Change the number of subunits to 2, and type in the gene names HP1587 and
HP1588, respectively. After typing in a gene name, click outside the text box to
have the system process your input.
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When you click OK, the system assigns a name for the new complex. sometimes
it take a few seconds for the system to assign the name – don’t click any
button until the name shows up!
When you click OK this time, the Protein Editor for the new protein opens up.
Type “3-keto-L-gulonate kinase I”. When you click OK, an alert box shows up.
Why?
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Correct use of enzymatic names. Note that this name is not an acceptable
enzymatic activity name – the “I” at the end indicates a particular allele, and not a
general enzymatic activity. To fix this, click “Return to Protein Editor”.
Back in the editor, remove the “I” from the enzymatic activity name. however, you
can still give this name to the protein using the optional “Enzyme Name” field at
the top of the editor .Click the button “Edit Enzyme Name” and type the name “3keto-L-gulonate kinase I”.
4. Adding coefficients and names to subunits. Scroll down to the subunit
sections, and change the subunit names to “3-keto-L-gulonate kinase α
subunit” and “3-keto-L-gulonate kinase β subunit”. Add coefficients of 2 to
both subunits. Click OK, and you should now see the following diagram:
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5. Adding evidence codes and creating publication frames. Open the
complex in the Protein Editor, and add the evidence code “Assay of purified
protein” (EV-EXP-ISA-PURIFIED-PROTEIN in older versions of the software).
Type the citation Paul73 next to it.
As soon as you click out of that box, a window will pop up to alert you that
publication Paul73 was not found.
Click “Search or Create Publication Frame”.
When he publication editor appears, enter data to your enjoyment and click OK
Back at the Protein Editor, click OK. Click on the pathway link (under the
enzymatic activity section). Notice that this enzyme shows up in darker color than
the previous one. This indicates a presence of an evidence code.
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6. Exporting the pathway. Right-click the pathway name, and select “edit ->
Add Pathway to File Export List”. Then click “File -> Export -> Selected Pathways
to Lisp-format File”. Click the “Enter File” button and specify the path and a file
name for the exported file. Check the “Export Enzymes and Genes?” option
(assume you are exporting the pathway for inclusion in MetaCyc).
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