You and your post-doc are analyzing the results from an experiment your graduate
ID: 150860 • Letter: Y
Question
You and your post-doc are analyzing the results from an experiment your graduate student calculated. The experiment used a well know substrate and a possible inhibitor. The data revealed that the substrate is binding to the enzymes because a product is formed. The student analyzed the concentration of the inhibitor in the solution and found 99.9% was unbound. The student also determined that an enzyme-substrate was formed with no bound inhibitor. The post doc noticed that the Michaelis Mentel constant was low compared to other irreversible reactions seen in the past. Describe the concentration of free enzyme after the enzyme-substrate complex is formed and after the reaction is complete.
Explanation / Answer
The Km or Michaelis Menton constant is the substrate concentration at half the maximum velocity (Vmax). It is the substrate concentration at which half the active sites of enzyme are filled. It is independent of enzyme concentration and depends only on enzyme structure.
Irreversible inhibitors bind to the enzyme so that it cannot bind the substrate. Hence, no enzyme-substrate complex is formed.
However, in this experiment, enzyme substrate complex is formed. Hence, the inhibitor is not effectively not binding to the substrate. Hence, it a weak irreversible inhibitor
The Km is low. Hence, the affinity of the substrate for the enzyme is high. This indicates that most of the enzyme active sites are filled with substrate.
Only 0.1% of the inhibitor was bound to the enzyme and not available for catalysis. Hence, 99.9% of the enzyme concentration is free to be bound by the substrate (assuming the enzyme has one active site that can be bound of the inhibitor; on enzyme: one inhibitor molecule).
When the enzyme substrate complex is formed, the substrate binds to the active site of the enzyme. Hence, concentration of free enzyme present will decrease as substrate is occupying its active site.
Once the product is formed, the active site of the enzyme will be free as the substrate is no longer bound to the enzyme. The enzyme substrate complex forms the enzyme-product complex and then products dissociate, releasing the free enzyme. Hence, the concentration of free enzyme should be be reinstated and will be nearly 99% (as 0.1% enzyme is bound to inhibitor).
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