FRANK M. HUENNEKENS 



unless the enzyme is first digested with a proteolytic enzyme 

 (5,38). This would suggest that the coenzyme is either bound 

 by a peptide or ester linkage, or possibly enmeshed in a protein 

 cage. The nature of the protein groups involved in flavin 

 binding has been investigated by Theorell, who suggested that 

 both amino groups and aromatic hydroxyl groups were respon- 

 sible. Whatever the possible types of binding are for the cases 

 cited above, it seems evident that for certain flavoproteins, 

 notably D-amino acid oxidase, where the conjugated enzyme is 

 resistant to dialysis but readily dissociated upon exposure to acid 

 conditions, the binding cannot be covalent. 



From this discussion of various aspects of nucleotide coen- 

 zymes, it should be apparent why metal ions, anions, and other 

 cofactors or activators like cysteine, were rather arbitrarily 

 excluded from the present group. Cysteine, and other reducing 

 agents, probably keep labile sulfhydryl groups on the enzyme in 

 the reduced state (although glutathione has a special role in the 

 glyoxalase reaction and ascorbic acid is specific in the enzymatic 

 reduction of folic acid). The anions and the monovalent cations 

 (Na+, K+, NH4+) play an important, but obscure, role in main- 

 taining the integrity of certain enzymes, possibly by controlling 

 the degree of hydration around the protein. The other metal 

 ions can be divided into two separate categories. The first are 

 those such as copper, molybdenum, and iron which are them- 

 selves capable of oxido-reduction, and which have potentials 

 within the range of other oxido-reduction coenzymes. These 

 metals may often participate as regular electron carriers, 

 although in metalloflavoproteins they may be concerned, in 

 addition, with binding of FAD to the protein or to the reactants. 



The other metals, such as Mg++and Mn++, probably assist 

 the binding of substrate or coenzyme to the protein. At 

 physiological pH most substrates and coenzymes are anions and 

 where the "active center" of the protein may be deficient in 

 appropriate cationic groups, the metal can then serve as a 

 bridge between anionic groups of the coenzyme or substrate and 

 the protein. The almost universal requirement for Mg++ in 



514 



