The Catalytic Effect of Intermediates and Inhibitors on the Krebs Cycle
One of the important concepts established by many of Krebs' experiments was that the organic acids that are produced during the citric acid cycle can stimulate oxygen consumption. In addition, Krebs noted that these intermediates are generated in cyclical fashion and that each acid serves as the substrate for the next enzymatic reaction in the cycle. Therefore, once one organic acid is formed, the rate of subsequent reactions is dependent on the production of this organic acid. By following the production and catabolism of organic acids in the pathway it was possible to determine the sequence of enzymatic steps involved in the Krebs cycle.
The use of metabolic inhibitors was an essential aspect of the experiments which determined that the reactions of the Krebs cycle are cyclical. By adding an inhibitor and then measuring the accumulation of an intermediate, it can then be possible to determine the order or sequence of reactions in the cycle. The following assignment is designed to help you determine the sequence of reactions involved in the Krebs cycle by studying the effects of inhibitors on oxygen consumption.
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 by adding mitochondria and malonate. Allow the reaction to proceed for one minute then add succinate. What happens to oxygen consumption in the experiment? 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. Consult a textbook if needed to verify your answer and to identify the enzyme inhibited by malonate.
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?
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