COENZYMES DERIVED FROM B VITAMINS 145 



be prepared synthetically, it can be obtained only by direct isolation from 

 tissues or yeast, or by separating it from preparations of its flavoproteins. 

 The ability to synthesize the two coenzymes must be common to most 

 living organisms, since no instances are known in which either of the 

 intact coenzymes must be supplied to an organism. 



Properties of the Flavoproteins. When the riboflavin coenzymes com- 

 bine with their apoenzymes to form the flavoproteins, certain changes 

 can be detected in the properties of the pigment. The flavin no longer 

 exhibits its characteristic fluorescence, although its yellow color is im- 

 parted to the enzyme. 62 



The redox potential of the flavoprotein system is appreciably higher 

 than for the uncombined flavins, i.e., the flavoprotein has a greater tend- 

 ency to accept hydrogen atoms and become reduced. 62 



Several different lines of evidence indicate that riboflavin phosphate 

 is united to its apoenzyme through both the acid group of the phosphate 

 ester and the imide nitrogen of the isoalloxazine nucleus, presumably by 

 the formation of salts with acidic and basic groups of the protein: the 

 affinity of unphosphorylated riboflavin for the protein is small; the sub- 

 stitution of groups upon the imide nitrogen destroys all vitamin and 

 coenzyme activity; the formation of salts of the imide is known to destroy 

 the fluorescence of isoalloxazines; and the flavin loses its fluorescence 

 when it combines with apoenzymes to form the flavoproteins. 



Unlike the nicotinic acid coenzymes, the flavin coenzymes do not have 

 to alternate from one apoenzyme to another during the progress of a 

 reaction. Hence, (in neutral solutions) the riboflavin enzymes are only 

 slightly dissociated. For this reason, when the "old yellow enzyme" is 

 resynthesized from its resolved parts, the apoenzyme takes up the added 

 riboflavin phosphate in stoichiometric fashion and is completely saturated 

 by the time an equimolecular amount of coenzyme is added. Flavoproteins 

 are not readily resolved by dialysis in neutral solution, although they are 

 easily dissociated in acidic solutions in which the pigment dialyzes away 

 from the protein. 65 A simpler procedure is to salt out the protein from 

 the acid solution leaving the prosthetic group in the supernatant liquid. 66 

 This difference in the stability of the intact enzyme under neutral and 

 acidic conditions is probably due to ionic and tautomeric changes in the 

 groups of the coenzyme and the protein which form the salt linkages. 



In addition to the differences in motility of their respective coenzymes, 

 there are a number of other dissimilarities in the enzymatic reactions 

 catalyzed by flavoproteins and those catalyzed by the nicotinic acid sys- 

 tems: (1) a considerably smaller number of reactions have been charac- 

 terized for the flavoproteins; (2) there is much more specificity in the 

 coupling of the two systems which oxidize and reduce the flavoproteins 



