Materials and Procedures

Materials

  • A computer with internet access.

Procedures

Record your answers to the following prompts in your notebook as you gather background information for the Lab 4 Assignment and future tyrosinase assignments. When you are finished, your instructor/TA will check your responses and sign you out only if you have completed all of these exercises. Feel free to follow your curiosity and ask questions if you are stuck.

  1. In one sentence, write the original research problem that you will investigate in with the class activity data. In a second sentence, explain how the data you have available will be used to answer/solve the question/problem that you pose.

    • Use online bioinformatics tools to answer the following questions about the enzyme tyrosinase.
    • If you type the name of the database into any search engine, the first hit is usually the correct link.
    • Remember to obtain original citations that you will use in your bibliography.
    • Click this link for a walkthrough of screenshots from each database

The NCBI Protein Database

Visit NCBI to identify Agaricus bisporus tyrosinase isozymes. 

  1. Use the Protein database to record the number of Agaricus bisporus tyrosinase isozymes that have been verified in the literature. Record the amino acid length of each.

    • To narrow your results by species, you can either use filters or advanced search. To filter, search for the enzyme in the in the main search prompt, select Fungi under the Species heading on the left and find Agaricus bisporus under Top Organisms on the right. To use advanced search, press Advanced under the search bar; in the Builder type the enzyme name under All Fields in the first prompt; in the second prompt select "AND" "Organism" then enter "Agaricus bisporus".

    • Narrow down your search to UniProtKB/Swiss-Prot. This eliminates redundant entries and shows you just the known isozymes categorized by number. Note that these isozymes are named PPO1, PPO2, etc. Use these names in your notes to identify them.

  2. Isozymes are enzymes that differ in amino acid sequence but catalyze the same chemical reactions and present in the same individual. Isozymes are expressed by completely different genes, have different maximal activity, and different binding properties with substrates.

    Let's observe the sequence differences between tyrosinase isozymes. Select "Align sequences with COBALT" near the upper right corner of the page displaying your hit results. Then click the Align button on the next page. When the results appear, change the "Conservation Setting" to "Identity". You will see a structural alignment of the isozyme sequences. Some of the isozymes will have extra loops of sequence and will cause gaps. Red amino acids are identical in all of the isozymes. Blue amino acids vary between isozymes.

    It is not currently known whether isozymes are expressed at different levels in different tissues or if multiple isozymes are even present in the same tissue of A. bisporus. Therefore, it is unclear how much the trends you will describe in the Lab 4 assignment are due to different enzyme levels or different isozymes of tyrosinase.

    Answer these questions: When you mention that mushroom tyrosinase has isozymes in your Discussion for the Lab 4 Assignment, what will you say about the impact of isozymes on your ability to interpret your results? What experiment(s) could help you identify the presence of isozymes?

The Protein Databank

Visit the PDB to gain insight into tyrosinase structure and mechanism.

  1. Search for structures of tyrosinase. Refine your search by selecting "Agaricus bisporus" under "Scientific Name of Source Organism" on the left bar and then press the green arrow next to the word "Refinements".

    Select one A. bisporus tyrosinase structure that has an inhibitor bound to the active site. (a) State the four-character PDB ID code for the structure. Read the PubMed abstract in the PDB entry. (b)  Describe at least two interesting features that the authors note about their structure. For example, is the structure a multimer (e.g. tetramer) of many proteins, is an oxygen atom bound, is the structure in the latent of activated form?

  2. Under "3D View" on the left of the page, open the "Ligand Interaction" display. (a) Identify the types ions and number of heavy metal ions that are bound to the active site. When you click on any ligand, dashed lines will show you the non-covalent interactions that bind that ligand to the protein. (b) Describe the types of non-covalent interactions that bind the ions to the protein (the viewer identifies the bond type if you mouse over it). (c) List the amino acid side-chains that bind the ions to the protein.

    • At the top of the viewer you will see "1: Tyrosinase". This shows you which sequence features are displayed. Click this to get a drop-down menu and select a metal. Now the metals are available in the sequence view. Click one to zoom-in on it.

The Baunschweig Enzyme Database (BRENDA)

Visit the BRENDA Enzyme Database to learn about molecules that activate and inhibit tyrosinase.

  1. Search "tyrosinase". State the tyrosinase EC classification that is most relevant to the enzyme (not the reaction) you have been working with in lab. The EC classification you select should give the most results that include Agaricus bisporus tyrosinase. Refer to the background of Lab 2 if you are uncertain which search result applies.

  2. Explore enzymes in BRENDA with this EC number. In one or two sentences, describe the general reaction(s) carried-out by all enzymes with this EC number.

  3. Under "Enzyme-Ligand Interactions" select "Substrates/Products". Select any substrate that looks interesting to you and click the structure icon (structure) to display the chemical reaction of that substrate with tyrosinase. (a)  Draw the structures of the substrate and products for this reaction. (b) Using the language of the sentence(s) you just wrote, label the functional groups that changed in the reactant and product of your reaction. 

When you write the Introduction section for the Lab 4 Assignment, you will describe the general reactions of tyrosinase in your own words. If you still feel uncertain about precisely describing the tyrosinase reaction at this point, please ask for assistance from the TA or instructor before proceeding.

  1. Under "Enzyme-Ligand Interactions" select "Activating Compounds". (a) Relative to other tyrosinase activators, how much literature is there on SDS as a tyrosinase activator? (b) Locate a reference that specifically studies SDS activation of Agaricus bisporus tyrosinase. You should cite this reference in your Introduction section for the Lab 4 Assignment.

In the final two research activities you will obtain information for the Tyrosinase Report in which you will be studying the activity of catechol as a tyrosinase substrate and the effects of tyrosinase inhibitors.

  1. Under "Functional Parameters", locate a published KM value for the catechol with Agaricus bisporus tyrosinase. Record the value that has conditions most similar to the conditions of our kinetics assays (if the conditions are not reported in BRENDA, you must read the actual article to find the conditions in the procedure section). Report the KM value, the conditions, and the full primary literature reference. You should save this reference on your computer because you will need it for the Lab 5 Assignment.

  2. Under "Functional Parameters", locate a  Ki value for one of the following well-studied tyrosinase inhibitors: salicylhydroxamic acid (SHAM), tropolone, or kojic acid. You will study the inhibitor you select here in Lab 6. Click the structure icon (structure) to view its structure. Report the Ki value, the conditions of the experiment, and draw the structure.

 

Before you leave today...

  • Submit your responses to the above questions either physically or electronically to the Lab 4 Notebook Submissions folder (ask your instructor which they prefer).
  • Make sure that all of your group's data is calculated correctly and posted.
  • Make sure that you understand all instructor feedback on your graded writing to date.