Objective of the enzyme inhibitor lab
Determine if and how 3 different chemicals inhibit the enzyme
What was the enzyme used in the enzyme inhibitor lab?
Succinic dehydrogenase
What was the substrate used in the lab?
Succinic acid
What is the purpose of the substrate?
To activate the enzyme
Competitive Inhibitor (3)
They bind at the active site, same spot as the substrate wants to bind
They compete with the substrate for the active site
Inhibitor typically has a structure similar enough to the natural substrate to be recognized by and fit into the enzyme’s active site.
Non-competitive/allosteric inhibitor (2)
They bind not at the active site but at another site (the allosteric site). Results in a changed shape of the active site.
They inhibit by changing the shape of the active site
Structurally, which potential inhibitor has the most similar shape and charge to the substrate?
Malonic Acid
Similar shape, same charges on both ends of the molecule, both have COOH groups on the end
Substrate
Molecule that binds to an enzyme to start a chemical reaction
Enzyme
Catalysts that speed up a reaction
Needs a substrate to work
Competitive and Non-competitive inhibitors
Prevent a substrate from binding to an enzyme
They are not substrates themselves
Will the reaction take place under these conditions:
Competitive inhibitor
Normal Substrate concentration
Reaction will not take place
Will the reaction take place under these conditions:
Competitive inhibitor
High substrate concentration
Reaction will take place
There is enough substrate present to beat the competitive inhibitor to the binding site
Will the reaction take place under these conditions:
Non-competitive inhibitor
Normal substrate concentration
No
Non-competitive inhibitors do not compete for the enzyme’s active site.
They bind to a different, allosteric site on the enzyme, inactivating it or destroying its ability to catalyze the reaction, even when the substrate is still bound to the active site.
Will the reaction take place under these conditions:
Non-competitive inhibitor
High substrate concentration
No
Non-competitive inhibitors do not compete for the enzyme’s active site.
They bind to a different, allosteric site on the enzyme, inactivating it or destroying its ability to catalyze the reaction, even when the substrate is still bound to the active site.
Will the reaction take place under these conditions:
Not an inhibitor
Normal substrate concentration
Reaction takes place
Will the reaction take place under these conditions:
Not an inhibitor
High substrate concentration
Reaction takes place
Clear test tube means…
Blue test tube means…
Clear: Reaction took place
Blue: Reaction inhibited
Which one is the competitive inhibitor?
Malonic acid/Sodium malonate
Resembles the chemical structure of succinate by having COO- groups at each end
Which one is the noncompetitive inhibitor?
Methylparaben
Which one is not an inhibitor?
Acetic Acid
What is the purpose of a flow chart?
Helps imprint important steps and their sequence serves as a quick reference
A good flow chart should try to prevent inadvertent errors
What were the three potential inhibitors?
1) Malonate
2) Acetate/Acetic Acid
3) Methylparaben
What did we mix with the beef heart in the homogenizer?
Ice cold, 0.25 M sucrose solution
