202 A SYMPOSIUM ON RESPIRATORY ENZYMES 



involved in reaction 4. He oflFers several possible explanations for 

 these occasional failures, although the matter must still be con- 

 sidered unsettled. 



In terms of the postulated cycle, the possible participation of 

 intermediate steps such as those listed in equations 1 to 5 can also 

 be critically examined from the standpoint of the minimum rates at 

 which they occur. This has aheady been discussed in considerable 

 detail elsewhere (9). If we assume that the citric acid cycle repre- 

 sents the entire respiratory process involved in the oxidation of 

 pyruvic acid in pigeon breast muscle, then the total oxygen uptake 

 of the tissue represents a total of five consecutive reactions of the 

 cycle. Under circumstances in which individual reactions of the 

 cycle are isolated— for example, when alpha-ketoglutarate is added 

 to the malonate-poisoned tissue— the rate of oxygen uptake for the 

 conversion of alpha-ketoglutarate to succinate cannot be less than 

 one-fifth that for pyruvate oxidation. If the reaction velocity is below 

 this minimum requirement, the step can be excluded as an inter- 

 mediate in the overall reaction. On the other hand, if the isolated 

 reaction proceeds with a velocity greater than the minimum rate, 

 it may still be considered as an intermediate, since in the intact 

 system the concentration of the particular enzyme concerned may 

 exceed the quantity of the substrate normally available. When the 

 reactions summarized in equations 1 to 5 are examined from this 

 viewpoint, we find in each case that the rate of the isolated step 

 proceeds with the necessary velocity consonant with its composing 

 part of the cycle. 



The existence of what I have termed the Krebs reaction is gener- 

 ally accepted: most critical comment on the citric acid hypothesis 

 centers around the postulated intermediate foi*mation of citric acid. 

 It has been argued that failure of this substance to accumulate in 

 large amounts during pyruvate oxidation in various tissues is con- 

 trary to its postulated intermediate role. However, the accumulation 

 of an intermediate must represent a balance between its synthesis 

 and its removal, and the accumulation of large amounts of citric 

 acid would be much more difiicult to reconcile with the premise 

 that it is an active intermediate substance than would the fact that it 

 accumulates to only a limited extent. In addition, Krebs has demon- 

 strated an anaerobic reaction between citrate and oxalacetate to yield 

 alpha-ketoglutarate and malate (equation 9). Although there is 

 reason to question the validity of such anaerobic experiments as the 

 basis for assigning the role of hydrogen carriers to the oxalacetate- 



