544 2. ANALOGS OF ENZYME REACTION COMPONENTS 



of glutamate racemase by riboflavin is reduced by FAD. It is clear that 

 there is not much information on the inhibition of enzymes by riboflavin 

 analogs, especially by their phosphates, or FAD analogs. The mechanism 

 for the inhibition of enzymes in which the flavin coenzyme is tightly bound 

 is not known. However, it is possible to suggest three mechanisms. It is 

 now known that various flavins and their nucleotides form molecular com- 

 plexes with one another, and the formation of such complexes with bound 

 FMN or FAD may occur, preventing the normal interactions of the coen- 

 zymes in oxidation. In some instances the inhibitors may interfere with 

 the experimental electron acceptor, particularly when this is a dye. Lastly, 

 one must consider the possibility of nonspecific binding of these polyhetero- 

 cyclic compounds to the enzymes; one might predict that a number of en- 

 zymes not involving flavins would be inhibited by such analogs, but few 

 have been examined. None of the riboflavin analogs in Table 2-33 is a po- 

 tent inhibitor and it is unlikely that these inhibitions are responsible for 

 any of the in vivo effects observed. 



The coenzyme of the old yellow enzyme is FMN, and riboflavin-5'-sulfate 

 does not interfere with its binding to the apoenzyme, which Theorell et al. 

 (1957) explain by the less negative charge on the sulfate group. Riboflavin- 

 5'-sulfate, however, inhibits D-amino acid oxidase (Egami and Yagi, 1956), 

 so that the structural requirements for binding must be different in these 

 two enzymes. Yagi and Nagatsu (1960) have studied the effects of ribofla- 

 vin-5'-sulfate on rat liver mitochondrial oxidations of a-ketoglutarate, suc- 

 cinate, malate, and D-alanine, and found that no inhibition is exerted at 

 0.1 mM, which they interpret as due to the tight binding of the FAD in 

 the mitochondria. Aged mitochondria are stimulated by FAD and here in- 

 hibition by riboflavin-5' -sulfate can be demonstrated. The FAD analogs of 

 the various flavins have not been studied often but Huennekens et al. 

 (1957 b) found lyxoflavin-5'-phosphate to be active in the NADPH-cyto- 

 chrome c reductase (although less than FMN) and lyxoflavin dinucleotide 

 to be active in the D-amino acid oxidase (but less than FAD). We have 

 seen that riboflavin deficiency leads to reduction in the activities of certain 

 enzymes in the tissues. Administration of galactoflavin to rats for 15-28 

 days leads to an approximately 40% reduction in glutamate and /5-hy- 

 droxybutyrate oxidation in liver mitochondria, but no change in succinate 

 oxidation or in the P : ratios (Beyer et al, 1961). U-2113, the 9-hydrox- 

 yethyl analog of riboflavin, causes a slight (15%) decrease in tumor xan- 

 thine oxidase in mice. It is not known if this is due to FAD depletion or a 

 more direct inhibition. 5-Hydroxytryptamine (serotonin) is metabolized by 

 monoamine oxidase to 5-hydroxyindoleacetate; both riboflavin deficiency 

 and galactoflavin increase the urinary excretion of this product, indicating 

 that one of the other metabolic pathways for serotonin is depressed by 

 interference with flavin function (Wiseman and Sourkes, 1961). These mis- 



