Assignment 1

1 .		Malonate is an analog of one of the organic acids that is naturally produced during the Krebs cycle. Malonate acts as an inhibitor of the Krebs cycle. Your job is to determine which enzyme in the cycle is inhibited by malonate. Begin an experiment in MitochondriaLab by adding mitochondria and malonate. Allow the reaction to proceed for one minute then add succinate. What happens to oxygen consumption in the experiment?
2 .		To determine which step in the Krebs cycle is inhibited by malonate, try adding pyruvate to the experiment. Perform similar independent experiments by adding malate, fumarate, and ADP, to mitochondria containing malonate. Try other substrates as well. For each experiment describe what happens to oxygen consumption. What do these result tell you about the sequence of succinate, pyruvate, malate and fumarate relative to where malonate is acting? Based on your results, propose a order of reactions in which each substrate appears and suggest a step in the Krebs cycle that is inhibited by malonate.
3 .		Once you have determined malonate's site of action, explain what would happen to the concentration of each of the following molecules: citrate, isocitrate, a-ketoglutarate, succinate, oxaloacetate, malonate, ADP, and ATP in the experiment if you treated these mitochondria with excess amounts of malonate?