432 THIAMINE 



As we have not at our disposition pure, crystalline enzyme preparations, 

 the activity has to be related to one mole of the coenzyme, the thiamine 

 pyrophosphate. Peters and his collaborators stated that the activity of the 

 carboxylase in yeast and that of the pyruvodehydrogenase in brain disper- 

 sions were of the same order. The pyruvodehydrogenase at' 30° catalyzes 

 the uptake of 1500 moles of O2 per minute, computed per mole of thia- 

 mine pyrophosphate. 



Some years later. Green et al.,^^ and almost simultaneously Kubowitz 

 and Liittgens,^^ using a fairly purified enzyme preparation from yeast, 

 found that this carboxylase preparation catalyzes the decarboxylation of 

 900 and 700 moles of pyruvic acid at 30° and 20°, respectively, per minute 

 per mole of thiamine pyrophosphate. 



On the whole, the purity of the pyruvodehydrogenases from animal tis- 

 sues is less. It is difficult to obtain a solution free from cell particles. There- 

 fore we do not know whether the oxidative carboxylations are brought about 

 by enzymes that contain only thiamine pyrophosphate -f- magnesium as 

 the coenzyme part, or whether these enzymes contain also another oxidative 

 coenzyme. 



Green et al.^^ and Stumpf et at.- obtained pyruvodehydrogenase prepara- 

 tions that were free from flavin adenine dinucleotide and from cytochrome c. 



Stumpf et at. could demonstrate that pyruvic and a-ketoglutaric oxidases 

 from pigeon breast muscle were different enzymes. The most cogent evi- 

 dence came from summation experiments: by adding a-ketoglutaric acid 

 to a pyruvodehydrogenase preparation that was saturated with pyruvic 

 acid, an extra carbon dioxide production was obtained. 



Table I indicates that many different reactions are catalyzed by thiamine 

 pyrophosphate enzymes and that most of them attack pyruvic acid or 

 other a-keto fatty acids. 



It may be remarked that pyruvic acid stands at the crossroads of the 

 carbohydrate metabolism; it is the end point of the anaerobic processes, 

 and the starting point of the decarboxylation and oxidation reactions and 

 of the reversal of the anaerobic chain of reactions. This attack on pyruvic 

 acid may be a simple decarboxylation, an oxidative decarboxylation, a 

 carbon dioxide fixation, a dismutation, or a condensation. We may assume 

 that the exact type of reaction depends upon the protein, the apoenzyme, 

 with which the thiamine pyrophosphate is combined. 



In the experiments with tissues or crude extracts, the enzyme system con- 

 tained several other enzymes and catalytic substances, such as the C4- 

 dicarboxylic acids, besides the carboxylase. In their initial work Long and 

 Peters'^^ concluded that there is a distinct difference between the systems 

 in yeast and in the animal tissue. The yeast system decarboxylates only 

 the pyruvic acid, Avhereas in the tissue oxidation accompanies decarboxyla- 



