118 THE BIOCHEMISTRY OF B VITAMINS 



and reduction of the coenzymes. When the coenzyme is reduced, the 

 quaternary ammonium ion in the pyridine ring is converted to a tertiary 

 amine. This elimination of a strongly basic group increases the acid prop- 

 erties of the coenzyme. Since the reduced acidic compound may dissociate 

 more readily, it leaves the apoenzyme and is free to transport the hydro- 

 gen atoms to another enzyme system where these atoms will be passed on 

 to another substrate. Other processes which involve shuttling atoms or 

 reactive groups from one enzyme system to another may likewise function 

 because of differences in the degree of dissociation of the two forms of a 

 coenzyme. 



The association of a substrate with its enzyme probably is always 

 accompanied with a redistribution of intramolecular energies within both 

 the substrate molecule and the protein. These changes are responsible for 

 the "activation" of a certain atom or group of the substrate molecule. 

 When a coenzyme unites with a protein the same thing occurs. The effect 

 is more than just the tying together of two substances, and even though 

 the union may be a "loose" one it results in profound changes in some of 

 the chemical properties and reactivity of both the coenzyme and the 

 protein. 



An interesting example in which a quantitative expression can be 

 derived for the change that takes place within a coenzyme when it com- 

 bines with a protein is riboflavin phosphate. The standard redox potential 

 of the riboflavin coenzyme is considerably different from the potential of 

 a riboflavin enzyme system. 24 This means that in combining with the 

 apoenzyme some changes take place within the riboflavin moiety which 

 greatly increase its tendency to accept hydrogen atoms (actually elec- 

 trons, p. 128) . The magnitude of the changes is such that the system can 

 catalyze an entirely different group of biological oxidations and reductions 

 than it could if it retained the potential of the unassociated coenzyme 

 (p. 146). 



The changes in the stability of apoenzymes when dissociated from their 

 coenzymes can be used to substantiate the fact that the separation of a 

 coenzyme from its protein may induce critical changes within the protein 

 molecule. For example, when the coenzyme is removed from a-amylase 

 by dialysis, the protein rapidly deteriorates and cannot be reactivated 

 again, even by the addition of the coenzyme. However, it can be shown 

 that the denatured protein still possesses its original capacity for com- 

 bining with the coenzyme (p. 125), although the complex formed is 

 enzymatically inactive. This indicates that in the dissociation of the 

 holoenzyme an alteration in the molecular structure of the protein occurs 

 at some position other than the exact site where the coenzyme attaches 

 itself. 



