BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 119 



The association of a coenzyme with its apoenzyme to form the holo- 

 enzyme is sometimes an essential step in the biosynthesis of a coenzyme. 

 Thus, the enzymatic synthesis of cocarboxylase from thiamine requires 

 the presence of the apoenzyme, and as soon as there has been sufficient 

 synthesis to saturate the protein the synthesis of the coenzyme ceases. 10 

 A reasonable explanation for this is that, when there is no longer any 

 apoenzyme present to combine with the coenzyme as it is formed, the 

 uncombined coenzyme cannot protect itself from the action of phos- 

 phatases which hydrolyze the coenzyme as rapidly as it is formed. 



Specificity of Coenzymes. The inactivity of most hydrolytic and other 

 degradation products of coenzyme molecules indicates that the structural 

 specificity required by apoenzymes of their coenzymes usually applies to 

 the whole coenzyme molecule rather than to one particular type group 

 or linkage within the molecule. The specificity of some apoenzymes for 

 their coenzyme cannot be said to be absolute, however. The well sub- 

 stantiated cases in which an analogue of a vitamin has vitamin-like 

 activity (Chapter VID) necessitate assuming that coenzymes containing 

 those vitamin analogues are synthesized enzymatically, and that the re- 

 sulting analogues of the natural coenzymes are able to combine with the 

 apoenzymes and catalyze certain reactions. A few analogues of coenzymes 

 have been prepared synthetically and tested in vitro and found to have 

 some coenzymatic activity. 



Analytical Methods for the Coenzymes. It would be gratifying in a 

 general discussion of the analytical methods for determining the coen- 

 zymes (and the enzyme systems in which they participate) to be able to 

 describe general methods which could be used for all the B vitamin coen- 

 zymes. Unfortunately, this is not possible at the present time. Studies of 

 the distribution of the B vitamins in cells and tissues have yielded some 

 very interesting results (Chapter II A). Such data would have consider- 

 ably more significance if they indicated how much of the vitamin was 

 in an active form and how the coenzyme was distributed among the 

 various systems for which it is required. 



It may be that microbiological methods, as general in their applicability 

 as the vitamin assays, can eventually be developed for the coenzymes. 

 This will depend upon success in finding organisms which require specif- 

 ically the intact coenzymes. There are no organisms known which satisfy 

 this requirement in the strictest sense, the closest case being an organism 

 whose vitamin B 6 requirement can be met only by pyridoxal phosphate 

 or pyridoxamine phosphate. 20 If suitable organisms cannot be found, 

 inhibitors perhaps will offer the solution to the problem of obtaining 

 simple general methods for the analyses of the coenzymes. The ideal 

 inhibitors for these analyses would effectively block the last reaction in 



