Escherichia coli (LacZ)
Beta Galactosidase

As you proceed through this tutorial buttons must be activated in sequence to work correctly.  If you want to redo the tutorial RELOAD the program.

I. Introduction

Beta galactosidase the molecule rotating on the left is an enzyme that initiates the breakdown of the sugar  lactose.  Lactose is a type of sugar found in milk.  It is composed of two  rings.  The rings contain 5 carbon atoms, one at each corner  (not shown) and an oxygen atom (O).  Attached to the carbon atoms are a hydrogen atom (not shown --) and an OH group or another carbon (C).

The rings are bound together by an oxygen bridge. This bridge can be broken when the enzyme beta galactosidase binds to lactose and a water molecule reacts with the oxygen atom in the bridge.  Reactions in which water molecules are added are known as hydrolysis reactions.

The beta galactosidase you will be using in lab this week was isolated from the bacteria Escherichia coli.  This bacteria produces beta galactosidase when lactose is present in its growth media.

II. General Structure of Beta Galactosidase Complexed with Lactose

The structure of the molecule on the left was determined using X-ray diffraction techniques.  It is a homotetramer meaning that it is composed of four identical chains of amino acids.  Click the button to more clearly see each chain.    <>  Each chain has a site where it can interact with a lactose molecule.  Click the button to see how the lactose molecule associates with the enzyme.  <>   If you right click on the molecule with your mouse you will bring up a menu that will allow you to stop the rotation of the molecule.  Then by left clicking and holding the button down you can rotate the molecule to more clearly see the lactose as it associates with the enzyme.  Try to orient the enzyme lactose complex so that you can see the double ring structure of the lactose.  Zoom in on the lactose molecule. <>  In this view you are only seeing the backbone of the homotetramere and the structure of the lactose molecule.   You may rezoom if you like.

III.  The Hydrolysis Reaction

Association of lactose with beta galactosidae facilitates the reaction between water and lactose.  This breaks the oxygen bridge and results in the production of two simple sugars.

IV.  The Assay

In your experiments you will be using ONPG in place of lactose.    Like lactose ONPG is a molecule composed of two rings held together by an oxygen bridge. Beta galactosidase also forms a complex with ONPG  molecules and is able to hydrolyze them. Load the beta galactosidase ONPG complex Be patient as it will take awhile for the molecule to load.   ONPG occupies a position similar to that of  lactose.  To see the ONPG molecules click on the button <>.   In this view molecules associated with the protein chains are shown in a space filled form.  ONPG appears as a large cluster of molecules embeded in the four protein chains.  You can also see the  sodium  and magnesium atoms associated with the protein backbone. Activate this button to more clearly see the ONPG <>  Right click on the molecule and stop the rotation.   Now hold the left button down and rotate the molecule to see the location of the ONPG molecules.  They are composed of double rings just like lactose.  How many ONPG molecules are complexed with each chain?

Beta galactosidase facilitates the hydrolysis of ONPG.  When ONPG is hydrolyzed a yellow product ONP is produced.  The intensity of color produced can be used to measure the rate at which beta Galactosidase can hydrolyze ONPG (or lactose).