COENZYMES DERIVED FROM B VITAMINS 157 



of cells. The results of studies of the biological synthesis indicate that 

 adenosine triphosphate is probably the usual phosphorylating agent. 122 * 123 

 It is needed in only catalytic amounts, however, if other acyl phosphates 

 are supplied or if the biosynthetic reaction is coupled with enzymatic 

 processes in which reactive phosphate derivatives are created. A cell free 

 extract of rat kidney which converts thiamine to its coenzyme has been 

 reported. 124 



The pyrophosphate linkage in the coenzyme is readily hydrolyzed by 

 the phosphatases (distinct from the phosphorylases catalyzing its syn- 

 thesis) and usually any excess coenzyme present in a cell will be rapidly 

 hydrolyzed. 114 If, however, the coenzyme is combined with apocarboxylase 

 it is quite resistant to attack by any hydrolytic enzymes present. This 

 may be one explanation for the fact that the synthesis of the coenzyme 

 from thiamine in a number of organisms is observed only as long as 

 uncombined apoenzyme is present. 



Mention has already been made of the inhibition of thiamine pyro- 

 phosphatases by thiamine and its derivatives. This type of inhibition has 

 been observed only in certain yeast, however. It probably accounts for 

 the exceptional case in which considerable unbound coenzyme can be 

 found in yeast cells. If these particular yeasts are cultured in media 

 containing appreciable amounts of thiamine, they absorb the vitamin 

 almost quantitatively and convert most of it to the pyrophosphate (in 

 amounts much greater than could be bound to carboxylase apoen- 

 zymes). 125 The coenzyme synthesized by these cells is not hydrolyzed, 

 since their specific phosphatases present have been inactivated by the 

 high thiamine concentrations. 



The pyrophosphate linkage between the two phosphate residues is also 

 readily hydrolyzed by dilute acid. The monophosphoric acid ester formed 

 by either the acid or enzymatic hydrolysis of the coenzyme is much more 

 slowly attacked by phosphatases. The presence of appreciable quantities 

 of the monophosphoric acid ester of thiamine in natural extracts probably 

 is the result of the breakdown of the coenzyme during the preparation 

 of the sample. 



The mechanism for the formation of holoenzymes from thiamine pyro- 

 phosphate and its apoenzymes has been the subject of several studies. The 

 easily prepared apocarboxylase from yeast has been used as the protein 

 source for the most detailed investigation. 126 The presence of the Mg ++ 

 or Mn ++ ion is essential for the union. Other divalent ions are much less 

 effective. In earlier reports it had been assumed that the artificially recon- 

 stituted holoenzyme was different from the original native system, since 

 the addition of a given amount of thiamine pyrophosphate to washed 

 yeast cells did not elicit as great a response as was obtained when un- 

 washed cells containing the equivalent amount of the coenzyme were 



