PROSTHETIC GROUPS, COENZYMES AND ENZYMES 



increasing the effectiveness of the colHsions with FMN mole- 

 cules, but other explanations are also possible. 



Some recent experiments confirmed the role of primary 

 amino groups (11) in a convincing way. 



Acetylation of a small percentage of the primary amino 

 groups of the apoprotein with acetic anhydride in acetate solu- 

 tions resulted in a strong reduction of the combination velocity. 

 If O.Y.E. is acetylated in the same way, the enzyme dissociates. 

 Neither of these effects is due to acetylation of thiol or tyrosine- 

 OH groups, and must therefore depend upon acetylation of 

 amino groups. 



Formaldehyde in water did not cause any rapid appearance 

 of free FMN from O.Y.E. This was expected, since formalde- 

 hyde reacts only with uncharged — NH2 groups, and in the O.Y.E. 

 the 



O— 



/ 



— p=o 



\ 



o— 



stabilizes the positive charge of the bound — NH3 groups. In 

 the presence of sodium chloride, however, formaldehyde in- 

 creased the dissociation velocity. Association experiments gave 

 very clear results: a decrease in kx was observed immediately 

 with formaldehyde concentrations as low as 0.02 M. Just as 

 might be expected, the inhibition increased with /?H. 



The strong and immediate effect of low concentrations of 

 formaldehyde suggests that the FMN-binding amino groups are 

 in an exposed position on the surface of the protein molecule. 

 This is not surprising, since it may be a prerequisite for their 

 ability to combine with FMN. 



The question arose which protein group is attached to the 

 imino group in the flavine ring system, thus quenching the 

 fluorescence. Weber (36) suggested it might be a tyrosine 

 hydroxyl group, without giving any experimental evidence in 

 this direction. We have experimental results which show that 



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