180 THE BIOCHEMISTRY OF B VITAMINS 



enzymatic biosynthesis of the coenzyme has not been critically studied 

 and is therefore still poorly characterized. 



The phosphorylated esters of the vitamin components are quite stable 

 and are not as readily inactivated by enzymatic hydrolysis as are the 

 coenzymes of other vitamins in which pyrophosphoric linkages occur. A 

 high degree of association between the coenzyme and its recognized 

 apoenzymes has been demonstrated. This affinity of the apoenzymes for 

 their coenzymes accounts for the difficulties encountered in trying to 

 resolve some of the holoenzymes by simple procedures; it may also be 

 an important factor in preventing the hydrolysis of the coenzyme by 

 intracellular phosphatases. 



The dissociation constant has been measured for a representative 

 enzyme of each of the three recognized types of pyridoxal-catalyzed 

 reactions. 201, 213 The three values have similar orders of magnitude 

 and are so small that the amount of uncombined pyridoxal phosphate is 

 negligible as long as there is even a small excess of an apoenzyme. The 

 affinity can also be demonstrated by the use of an inhibitor, an analogue 

 of the coenzyme. The effectiveness of the analogue in inactivating a 

 decarboxylase system depends upon the order in which the analogue and 

 the coenzyme are added to the apoenzyme. 214 If the coenzyme is added 

 first the analogue is quite ineffective as an antagonist, whereas if the 

 order of addition is reversed the analogue is an effective inhibitor. The 

 association is thus great enough so that the equilibrium between the 

 inhibitor-apoenzyme and coenzyme-apoenzyme systems is not readily 

 achieved, and in such an instance considerable time would have to elapse 

 before the ratio of inhibitor to coenzyme would be the factor determining 

 the degree of inhibition. 



Dialysis of fresh preparations of the holoenzyme does not accomplish 

 its resolution. The most effective means of securing the apoenzyme from 

 the cellular material of normal organisms, i.e., those supplied adequate 

 amounts of vitamin B 6 , is a process of aging. Storing the intact tissue 

 or extracts for a period of time results in a gradual inactivation of the 

 preparation and the release of the apoenzymes. 198, 199, 215 The process, 

 however, is not one of simple dissociation but rather one in which destruc- 

 tion of the coenzyme precedes the dissociation. Dissociation of the com- 

 plete enzyme is also achieved whenever the source of the enzyme is carried 

 through the number of fractionating procedures needed to effect a sub- 

 stantial concentration of the enzyme. 216 The extent of destruction of the 

 coenzyme during such separations has not been indicated. 



Reactions Catalyzed by Pyridoxal Phosphate. The chemical changes 

 catalyzed by pyridoxal phosphate may, on first inspection, appear to 

 have little in common since they represent extremely divergent types of 



