i 



436 THIAMINE 



acid hydrogenase. Utter and Werkman*^ showed that the apparently "hy- 

 droelastic" action of this extract 



CH3COCOOH + HoO -^ CH3COOH + HCOOH M 



was really a phosphoroclastic" split. Utter et al?^ with the aid of C'^ as a 

 tracer demonstrated that this reaction 



CH3COCOOH + H3P04^ CHsCOOCPOaH.) + HCOOH 



is reversible. This reversibility shows that a thiamine pj^rophosphate en- 

 zyme can catalyze a synthesis, as well as a breakdown, of pyruvate. 



Acetic acid is formed by the dephosphorylation of acetyl phosphate. 

 The acetyl phosphate that is formed by the oxidative decarboxylation of 

 pyruvic acid by the action of thiamine pyrophosphate is a higher reactive 

 compound. Lipmann was able to prepare acetyl phosphate synthetically. 

 However this product was inactive. So the enzymatic intermediate must 

 have a somewhat different structure. 



Most of the thiamine-enzyme preparations from animal sources are easily 

 sedimented, e.g., by centrifuging 30 minutes at 15,000 or 20,000 r.p.m. 

 (Ochoa'^** and Green et al}^). Some of the enzyme preparations from bacteria 

 seem to be more resistant to centrifugal force. Thus Silverman and Werk- 

 man^" state that the carboxylase from Aerohacter aerogenes remained in the 

 supernatant solution after centrifuging for 1 hour at 250,000 to 300,000 

 r.p.m. With a preparation from Proteus vulgaris, Stumpf-^ found that cen- 

 trifugation for 1 hour in a gravitational field of 9700g sedimented only a 

 small part of enzyme. However, centrifugation for 1 hour in a gravitational 

 field of lOOjOOO^' sedimented all active material. There seems to be a differ- 

 ence between the different enzymes. 



3. Inhibitors and Activators 



Most of the preparations show no inhibition by 3.3 to 10 X 10~^ M 

 iodoacetate, fluoroacetate, fluoride, or malonate (Green el al.^^ Green et 

 al.,-^ Stumpf,-^ and Stumpf et alP). Some preparations are more or less 

 inhibited by iodoacetate and fluoride. Thus the enzyme from Aerohacter 

 aerogenes is inhibited 85 % by 7 X 10~^ M iodoacetate, and 36 % by 5 X 

 10~^ M fluoride (Silverman and Werkman^"), and the diacetyl mutase from 

 pigeon breast muscle was inhibited 39 % by 3.3 X 10~^ M iodoacetate 

 {Green et at }^). 



Salts of heavy metals were found to be strong inhibitors for the cocar- 

 boxylase from yeast (Greened aL"). A ilf/10,000 concentration of AgNOs , 



71 M. F. Utter, C. H. Werkman, and F. Lipmanu, J. Biol. Vhem. 154, 723 (1944). 



