IV. nUirHEMICAL SYSTEMS 433 



tioii. Animal tissues contain a |)>iu\ic acid dchydroj^onasc. J-'roni these re- 

 sults tlu'y inl'ciicd that the apoenzymcs arc (hlTcrcnt in yeast, and in animal 

 tissues. 



'I'lnis, thiamine pyrophosphate can act in yeast as the coferment of pure 

 carboxylase, whereas in animal tissues and in s(;veral l)acteria it acts as the 

 coferment of a pyru^'ic dehydrogenase. Watt and Krampitz, '''■''* using C'''02 

 with isotopic C as a tracer, were able to demonstrate that the transforma- 

 tion of pyruvic acid into a-acetolactic acid + CO2 is a reversible reaction. 



1. The Unitarian View 



Coenzymes commonly catal3''ze different reactions, depending upon the 

 nature of the apoenzyme. However, the difhculty with thiamine pyrophos- 

 phate is that the two kinds of reactions are so widely different from a chemi- 

 cal point of view — one is oxidative and the other is non-oxidative. Also, 

 none of the mechanisms proposed for the oxidative action of thiamine 

 pyrophosphate has proved to be satisfactory (see below). Krebs drew at- 

 tention to this difficulty, and he was the first who advanced an attractive 

 unitarian theory, bringing all the different activities of thiamine under one 

 single denominator. Evans,^" working in Krebs' laboratory, had shown 

 that minced pigeon liver was able to oxidize pyruvate to a-ketoglutarate. 

 From experiments to elucidate the mechanism of this reaction, Krebs and 

 Eggleston^^ were led to the assumption that the primary step is an assimila- 

 tion of carbon dioxide to pyruvic acid, a Wood and Werkman reaction.®^- ®^ 



CO2 + CH3COCOOH ^ COOHCH2COCOOH 



The oxalacetate formed in this way is converted into citrate and a-keto- 

 glutarate according to Krebs' citric acid cycle. Krebs and Eggleston as- 

 sumed that the first of this cycle of reactions, the formation of oxalacetate, 

 (the reverse of "decarboxylation") is catalyzed by thiamine pyrophosphate. 

 In this way, the \'itamin is not directly concerned with the oxidation of 

 pyruvate but with a reaction preliminary to the oxidation; the oxalacetate 

 acts as a hydrogen carrier. However, the experimental evidence for this 

 theory was not very convincing. Their chief argument was based on the 

 demonstration with suspensions of pigeon liver that pyruvate utilization 

 was greatly increased by suspending it in bicarbonate solution instead of 

 in phosphate buffer and by adding carbon dioxide to the gas phase, whereas 

 carbon dioxide and bicarbonate have no general effect on processes in liver 

 suspensions. 



"»D. Watt and L. O. Krampitz, Federation Proc. 6, .301 (1947). 

 «" E. A. Evans, Jr., Biochem. J. 34, 829 (1940). 

 «« H. G. Wood and C. H. Werkman, Biochem. ./. 30, 48 (1936). 

 «2H. G. Wood and C. II. Workman, Biochem. J. 32, 1262 (1938). 



