Malonate Inhibition of Oxidations in the Krebs Tricarboxylic Acid Cycle

The inhibition of succinate oxidation by malonate is a well known phenomenon.Since the oxidation of succinate to fumarate is an integral part of the Krebs cycle of oxidations, it has been generally assumed that the inhibitory effect of malonate upon the oxidation of any member of the cycle is the result of the inhibition of the succinate to fumarate step.However, the present paper provides evidence that malonate inhibits oxidations in the cycle by at least two mechanisms: in addition to the inhibition resulting from a block of succinate oxidation, malonate inhibits oxidation by another mechanism that is believed to involve combination with magnesium ions. MethodsThe reaction mixture has been described elsewhere (1).It contained (final concentrations) ~/15 KCl, ~/60 K phosphate at pH 7.2, 1.3 X lo+ M cytochrome c, and 0.001 M K adenosine triphosphate.The concentrations of MgClz, oxalacetate, pyruvate, K malonate, and rat tissue homogenate are given for the separate experiments.When oxygen uptake measurements were desired, the Warburg apparatus was used in the conventional manner at 38".Samples were equilibrated for 10 minutes and then readings were taken at 10 minute intervals.For some experiments substrate disappearance was considered a better measure of the reaction than oxygen uptake.When the rate of oxygen uptake was not required, samples were shaken in open flasks.At the end of the experiment 2 ml. of 17.5 per cent trichloroacetic acid were added to give a final concentration of 7 per cent; the precipitated protein was centrifuged and the supernatant fluid analyzed.All experiments were in duplicate, and the more important ones were carried out on two or more animals.The rats were all young adult males on a stock diet.Total keto acids were determined calorimetrically with 2,4-dinitro-