200 A SYMPOSIUM ON RESPIRATORY ENZYMES 



mating the physiological state, since they occur when pyruvate is 

 added to the tissue in the presence of an adequate supply of oxygen 

 and in a medium approximately physiological in ionic concentration 

 and pH. In these circumstances the stoichiometric relationships of 

 equation 7 are very closely realized, and it seems certain that the 

 total oxygen uptake of the tissue is utilized for the oxidation of 

 pyruvic acid completely to carbon dioxide and water. Pyruvic acid 

 will also disappear in the absence of oxygen. The quantity con- 

 cerned is about one-tenth that removed aerobically, and although 

 lactic acid, acetic acid, carbon dioxide, succinic acid, and beta- 

 hydroxybutyric acid have been recognized as products of this anaer- 

 obic reaction, the details are still obscure. 



Equations 1 to 5 deal with reactions carried out in the presence 

 of malonic acid. Equation 1 is the most important reaction of the 

 theory, since it demonstrates unequivocally an oxidative formation 

 of succinic acid from fumaric acid. In the presence of proper con- 

 centrations of malonic acid the oxidation of pyruvate by pigeon 

 breast muscle can be almost entirely inhibited. When fumarate is 

 added, however, pyruvate is oxidized and in accordance with equa- 

 tion 1 we find, per mole of fumarate added, one mole of pyruvate 

 and 2 moles of oxygen consumed, and 1 mole of succinate and 3 

 moles of carbon dioxide formed. A direct reduction of fumarate to 

 succinate under these circumstances is inhibited by the malonic acid 

 present, as can be shown by anaerobic experiments; that is, we find 

 much more succinate formed from fumarate in the malonate- 

 poisoned muscle in oxygen than in nitrogen. Since it is impossible 

 to explain the conversion of fumarate to succinate in the presence of 

 malonate by anaerobic reduction, a second mechanism must exist 

 which is oxidative and unaffected by malonate and which results in 

 the transformation of fumarate into succinate. It should be em- 

 phasized that we find in this transformation the stoichiometric re- 

 lationships of equation 1. The citric acid cycle postulates that this 

 oxidative transformation involves the intermediate formation of 

 citric acid. Regardless of the nature of the intermediates, however, 

 any explanation of the oxidation of pyruvic acid by muscle must 

 account for reaction 1, i.e., what might be teiTned the Krebs reaction. 



The demonstration of the stoichiometric relationships of equation 

 1 depends upon the recognition of essential experimental conditions. 

 In view of the fundamental nature of this reaction it may be per- 

 missible to discuss a few matters of experimental detail. 



The working plan of the experiments summarized in equations 1 



