Lab Concepts: Competitive Inhibition

In the Anatomy 36 lab on bioenergetics, you are asked to look at the effects of malonic acid as a competitive inhibitor of a reaction in which succinic acid is converted into fumaric acid with the help of the enzyme, succinic acid dehydrogenase.

In the normal reaction, depicted below, succinic acid (substrate) gives up 2 hydrogen molecules, which are transferred to the fumaric acid (product) by the energy transfer molecule, FAD+. The reaction is catalyzed by the enzyme succinic acid dehydrogenase (SAD), which is found in the ground beef.

COOH COOH | | CH2 CH | || + 2 H CH2 CH | | COOH FAD+ FADH2 COOH

Succinic acid fumaric acid (substrate) succinic acid dehydrogenase (SAD) (product)

SAD is an enzyme that only catalyzes the reaction that removes two hydrogens from the succinic acid (this is called a “dehydrogenation” reaction). To understand what happens next, it is important to remember that FAD+ is an energy transfer molecule. It only lasts a very short time and must offload what it has picked up as quickly as possible so that there are sufficient energy transfer molecules available for the transfers to continue as more reactions are catalyzed. In the illustration above, the FAD+ is reduced when it accepts the 2 hydrogens from the succinic acid, but it is immediately oxidized when it transfers those 2 hydrogens to fumaric acid.

Malonic acid is a substance that is very similar to succinic acid in structure. It is also capable of binding to the SAD, but when it binds, it binds with greater affinity, blocking the binding site and preventing it from acting on the succinic acid. With malonic acid blocking the active site of the SAD, dehydrogenation of succinic acid does not occur at a level that we can observe. (remember: enzymes never catalyze a reaction that wouldn’t occur anyway!).

COOH | CH2 | COOH No Hydrogen formed Malonic acid succinic acid dehydrogenase (SAD)

Methylene blue is an indicator that is blue when it is oxidized, and clear when it is reduced. If methylene blue is added to the first reaction, in which the substrate is succinic acid, the methylene blue will accept some of the H released by the action of the SAD and will turn clear. If it is added to the second reaction, methylene blue will be unable to accept any hydrogen atoms (there is almost no dehydrogenation), and it will remain blue. The reaction with the methylene blue indicator would look like this:

Methylene blue Methylene blue oxidized reduced

2 H COOH COOH | | CH2 CH | || + 2 H CH2 CH | | COOH FAD+ FADH2 COOH

Succinic acid fumaric acid (substrate) succinic acid dehydrogenase (SAD) (product)

+2H

Methylene blue (oxidized) Methylene blue (reduced)

FAD+ FADH2

-2H +2H

Succinic Acid Fumaric Acid

No Hydrogen produced

Malonic Acid (Inhibitor)

No hydrogen