444 THIAMINE 



The decarboxylation of a-ketoglutaric acid took place with the same 

 velocity as that of pyruvic acid. Green et al. made it probable that the 

 pyruvo(apo)carboxyla.se and the Q;-ketogiutaro(apo)carbox3dase were differ- 

 ent enzymes. For if thiamine pyrophosphate was split from the carboxylase 

 by washing at pH 4.6, the maximum rate for carbon dioxide development 

 with pyruvate as a substrate was found by the addition of 2 7 of thiamine 

 pyrophosphate per milliliter of Green's preparation. With a-ketoglutarate 

 as a substrate, the maximum reaction velocity was attained only when 300 

 7 of thiamine pyrophosphate per milliliter was added. A third enzyme 

 seemed to be present in Green's preparation, an enzyme for the condensa- 

 tion of acetaldehyde to acetoin. For maximum activity it required 30 7 

 of thiamine pyrophosphate per milliliter. Each enzyme perhaps has a very 



TABLE III 

 Decarboxylation of a-KETONic Acids by Yeast Carboxylase 



Substrate, 0.04 M CO2 , mm.' 



Pyruvic acid 596 



or-Ketoisovaleric acid 525 



Oxalacetic acid 191 



a-Ketoisocaproic acid 24 



«-Ketoglutaric acid 8 



Phenylpyruvic acid 



a-Hydroxyphenolpyruvic acid 



Acetoacetic acid 



No addition 



high specificity for different substrates and may be catalyzed b}' different 

 apoenzymes plus the same coenzyme. 



The pyruvic oxidase, prepared by Stumpf^ from Proteus vulgaris proved 

 to be very specific. It oxidizes only pyruvic acid; a-ketobutyric, a-ketocar- 

 poic, a-ketoglutaric, acetoacetic, glyoxylic, and lactic acids are not at- 

 tacked. The enzyme preparation oxidized formic acid and phenylpyru\dc 

 acids, even in the absence of thiamine pyrophosphate. Therefore, the oxi- 

 dation of these two acids cannot be attributed to the thiamine pyrophos- 

 phate-pyruvic acid oxidase. The diacetyl mutase preparation of Green et 

 al.'^ from animal tissues was found to be highly specific for diacetyl. Glyoxal, 

 methylglyoxal, dipropionyl, benzil, cyclohexamedione-1 ,2, alloxan, and 

 cyclohexanehexone were not attacked by the enzyme. However, the same 

 preparation catalyzes the oxidative decarboxylation of pyruvic acid and 

 of or-ketoglutaric acid (Stumpf et al}^). Here again, the a-ketoglutaric oxi- 

 dase, after splitting off the thiamine pyrophosphate, required a larger 



i« V. K. Stumpf, K. Zarudiiaya, and D. E. Green, ./. Biol. Chem. 167, 817 (1947). 



