136 1. MALONATE 



block it induces. The situation is vey similar to that of ghicose oxidation in 

 this respect. Generally speaking, malonate could act on either the cycle, 

 or the helix, or both. Despite the extensive work that has been done on the 

 effects of malonate on fatty acid oxidation, direct information on the actions 

 on the helix is lacking, since the five reactions involved in each turn of the 

 helix and the enzymes associated with these have not all been tested for 

 susceptibility to malonate, nor has the operation of the helix dissociated 

 from the cycle been studied. Our evidence on this point must be indirect. 



Before considering this evidence, let us outline some of the possibilities 

 for mechanisms of helix inhibition. Just as in the oxidation of glucose, ATP 

 is required for the initiation of the helix reactions and Mg++ is a necessary 

 cofactor (e.g., for the fatty acid thiokinase), so that malonate might depress 

 the operation of the helix by depleting the system of either of these sub- 

 stances. The extent of such an inhibition will depend on the availablity of 

 ATP or the presence of systems generating it, and on the concentration of 

 Mg++. Possibly a more important factor is the requirement for coenzyme A. 

 Malonate could deplete conzyme A by at least two mechanisms. The for- 

 mation of a relatively stable malonyl-CoA would remove some of the 

 coenzyme A from participating in the helix. A block of the cycle would 

 impede the entrance of acetyl-CoA into the cycle and the regeneration of 

 coenzyme A will depend on the enzymes present for the metabolism of 

 acetyl-CoA. The usual pathways for acetyl-CoA are (1) a simple splitting 

 to form acetate, (2) a transfer of the coenzyme A to another acid, and (3) 

 a condensation of two acetyl-CoA's to form acetoacetyl-CoA and eventually 

 acetoacetate. As in the oxidation of pyruvate through the cycle, the fate 

 of acetyl-CoA will depend also on the presence of reactions forming oxal- 

 acetate by pathways other than the cycle. The rate of fatty acid oxidation 

 can thus be limited by the rate of regeneration of coenzyme A. These 

 considerations lead one to ])redict that the effects of malonate on fatty 

 acid oxidation would be variable and dependant on the metabolic charac- 

 teristics of the tissue studied and the conditions of the experiment. This 

 prediction is borne out. 



There is a good deal of evidence that malonate in concentrations up to 

 20 raM does not directly inhibit the reactions of the helix. Although an 

 inhibition of the oxygen uptake or the CO2 production during fatty acid oxi- 

 dation is not indicative of the site of inhibition when the helix and the 

 cycle are operating together, the absence of inhibition implies a lack of 

 action on the helix. Malonate at 16.8 mM has no effect on the C^^Oa arising 

 from palmitate-1-C^* in soluble extracts of peanut cotyledons (Castelfranco 

 et al., 1955), nor does 1 roM malonate have an effect on the oxygen uptake 

 associated with palmitate oxidation in peanut microsomes (Humphreys 

 et al., 1954). The anaerobic dehydrogenation of C4-C18 fatty acids in liver 

 homogenates with methylene blue as an acceptor is not inhibited by 



