In this exercise you will use Chime to look at the active site of Carboxypeptidase A with and without a substrate. You must be using a Netscape browser (v3.x or above) and have the browser configured to use the Chemscape Chime plug-in.
The tutorial is divided into two parts. In Part One, you will isolate the active site of CPA with no substrate present. In Part Two, you will isolate the active site with the substrate Glycyl-Tyrosine in the active site.
You will have some questions to answer along the way! These will be indicated by the Q/A icon . When you see this icon, single click on it to go to the appropriate questions.
For further information on any of the commands used in this tutorial, consult the online RasMol Manual
You will be entering commands in the box just below the graphics window. Remember - you must select the command box (click in the window with the mouse) in order to type in the commands.
It is often more convenient to write a text file (known as a 'script' file) which contains the commands you wish to execute. The commands can then be executed all at once in RasMol using the 'script' command. However, because we are using the Chime plug-in for this tutorial instead of RasMol, we have placed buttons along the way which will execute the desired 'script' when you click on them.
As you proceed through the tutorial, click on the appropriate buttons to execute scripts. Click on the script name to view the script so that you are familiar with the commands executed within it (the commands will also be displayed in the Chime message box).
For example, the first script is called as1. It defines a set known as 'AS' to include members of the CPA active site. If you wish to use any of the scripts with RasMol, choose the link to the script files on this page and follow the instructions given.
The following commands should leave only the residues involved in the active site within the graphics window :
Now try zooming in on the active site. zoom 300 (enter)
Which amino acid residues are involved in the active site? First, let's pin-point the Zinc atom which is essential for enzymatic activity :
First, let's pin-point the Zinc atom which is essential for enzymatic activity :
Next, identify the residues which coordinate the Zinc atom :
Important Note! It is sufficient throughout this tutorial to enter the numbers of the amino acids without the three letter code. We will include them here for your information.
colour green (enter) <- Click to execute script as3 (enter) A script file labels the appropriate residues
Single click on the image if you wish to check your work.
Step Four - Experiment Experiment with the RasMol menu in the graphics window. First, select the active site : select AS (enter) Then try some of the Display options - eg: Spacefill and Colour options -eg: CPK Test your knowledge!. End of Part One PART TWO - The Active Site With Substrate (Glycyl-Tyrosine) Step One - Load Carboxypeptidase A plus Glycyl-Tyrosine <- Click on the button to load the Carboxypeptidase A + the Glycyl-Tyrosine substrate into the Chime Graphics Window. Step Two - Isolate the Active Site with Substrate You are now going to isolate the active site with glycyl-tyrosine as the substrate positioned within the active site.Once again, we have written a short 'script' file (as2) which defines a set known as 'AS' to include members of the carboxypeptidase A active site and the substrate glycyl-tyrosine.The following commands should leave only the residues involved in the active site within the graphics window.Commands: <- Click to execute script as2 restrict AS (enter) Step Three - Members of the Active Site Position the active site in the center of the graphics window. translate x -5 (enter) translate y -45 (enter) Now try zooming in on the active site. zoom 300 (enter) Which amino acid residues are involved in the active site? First, let's pin-point the Zinc atom which is essential for enzymatic activity : centre zn (enter) Molecular rotation now centred on the zinc atom. select zn (enter) <- Click to execute script cpa3zn (enter) The cpa3zn script uses the 'monitor' command to identify the atoms which coordinate zinc, note that the substrate displaces the water molecule in the active site. Next, identify the active site residues which coordinate the Zinc atom : select glu72, his69, his196 (enter) colour green (enter) <- Click to execute script as3 (enter) A script file labels the appropriate residues Finally, identify the amino acid residues which interact with the substrate : select arg127, asn144, arg145, glu270, tyr248 (enter) colour yellow (enter) <- Click to execute script as4 (enter) A script file labels the appropriate residues The remaining residues belong to the substrate. select substrate color cpk <- Click to execute script as5 (enter) A script file labels the substrate residues Which atom in the substrate becomes coordinated to the zinc atom? You may want to check this reference. Use the mouse to click on the zinc atom, then the atom in the substrate which coordinates the zinc atom (it may be easier to pick atoms if the substrate is displayed as a Ball and Stick model). Information about each atom is written into the command window: {atom} {atomno} {Type: residue} {residue no} [Chain: identifier] Notice that the second field contains the atom number. Use the monitor command to visualise the coordinating bond between the appropriate atoms, ie.: monitor atomno1 atomno2 Where atomno1 is replaced with the Zn atom number and atomno2 is replaced with the appropriate atom number from the substrate. Single click on the image if you wish to check your work. Step Four - Experiment Experiment with the RasMol menu in the graphics window. First, select the active site : select AS (enter) Then try some of the Display options - eg: Spacefill and Colour options -eg: CPK Test your knowledge!. Step Five - Active Site Environment You should be aware that carboxypeptidase A hydrolyses carboxy-terminal amino acid residues of proteins with bulky aliphatic or aromatic amino acid residues more quickly than it does other carboxy-terminal residues of proteins. You are now going to look at the environment about the amino acid side-chain of the substrate (phenolic group in this example of glycyl-tyrosine) to ascertain why this is so.Commands: select all (enter) wireframe (enter) select hydrophobic (enter) colour white (enter) select polar (enter) colour red (enter) select substrate (enter) colour green (enter) centre zn (enter) Position the substrate molecule so that the phenolic hydroxyl group of the substrate (in green) points away from you directly back into the screen. You may wish to use the rotate command. From the graphics window select sticks from the Display menu. Since the substrate is the last selected item, it should now be displayed as a stick structure. Let's now identify the amino acids that form a pocket to accomodate the side chain of the carboxy-terminal amino acid (which in this case is the phenolic group of tyrosine).Commands : define pocket 243, 250, 255, 203, 247 (enter) select substrate or pocket (enter) dots on (enter) The dot outlines show the force field of Van der Waals contacts (Be patient as it may take a few moments for the computer to calculate the field). You may wish to further isolate the substrate and it's 'pocket' with the following command: restrict substrate or pocket (enter) Test your knowledge! End of Tutorial
Test your knowledge!.
Once again, we have written a short 'script' file (as2) which defines a set known as 'AS' to include members of the carboxypeptidase A active site and the substrate glycyl-tyrosine.
The following commands should leave only the residues involved in the active site within the graphics window.
Commands:
Which atom in the substrate becomes coordinated to the zinc atom? You may want to check this reference. Use the mouse to click on the zinc atom, then the atom in the substrate which coordinates the zinc atom (it may be easier to pick atoms if the substrate is displayed as a Ball and Stick model). Information about each atom is written into the command window:
From the graphics window select sticks from the Display menu. Since the substrate is the last selected item, it should now be displayed as a stick structure.
Let's now identify the amino acids that form a pocket to accomodate the side chain of the carboxy-terminal amino acid (which in this case is the phenolic group of tyrosine).
Commands :
You may wish to further isolate the substrate and it's 'pocket' with the following command:
Test your knowledge!
. When you see this
icon,
single click on it to go
to the appropriate questions.