PROSTHETIC GROUPS, COENZYMES AND ENZYMES 



ki were determined both from the initial velocities at / = and 

 from different points on the curves before equilibrium was 

 reached ; K was, of course, determined from the amount of free 

 FMN at equilibrium starting from different concentrations of 

 O.Y.E. From dissociation experiments at 23.5° C, pH. 5.75, 

 0.4 M NaCl we found Arg = 13.5 X lO"" sec.-\ and K = 0.71 X 

 10~^. From these values 



1 ^ ^ V 1 ~4 



The direct determination of ki from the corresponding associa- 

 tion experiment was 



yti = 17 X IG^Af-^sec-i 



These experiments thus gave confirmation by kinetic methods of 

 the perfect reversibility of the reaction. 



INTERACTION OF RIBOFLAVIN WTTH THE APOPROTEIN 



In 1 936, Kuhn and Rudy (8) found that apoprotein and an 

 excess of riboflavin could substitute for O.Y.E. in the enzymatic 

 activity test. This observation made it likely that riboflavin 

 could form an active compound with the apoprotein, but ex- 

 perimental proof has been lacking. It was therefore of interest 

 to investigate whether the apoprotein could extinguish the 

 fluorescence of free riboflavin. This was found to be the case. 

 As seen from the lowest curve in Figure 3, ki is approximately 1 

 times lower than for FMN, and there is nothing which simulates 

 a dissociation curve in the pH region 4 to 8, just as would have 

 been expected. A maximum appears at pH 9.8, and ki de- 

 creases at higher pH values. The riboflavin-apoprotein com- 

 pound is much more dissociated than O.Y.E., mainly depending 

 on the fact that k2 is comparatively high, 1000 X 10~^ sec.~^ in 

 acetate /?H 7, where the k2 for O.Y.E. is certainly lower than 0.01 

 X 10~^ sec.~^ This illustrates the importance of the phosphoric 

 acid group for establishing functioning holoenzymes at reason- 

 able levels of coenzymes. 



289 



