PROSTHETIC GROUPS, COENZYMES AND ENZYMES 



Determinations of K with varied amounts of ADH and at 

 pH 6.4 to 10 revealed the fact that K was increased by a factor of 

 about 200, in the presence of a slight excess of ADH, in the pH 

 region 6.4 to 7.8. From pH 7.8 to 10 this factor decreased 

 gradually from 200 to 3. 



On the basis of the theories of Clark and his associates for 

 the redox potential of hemochromogens our findings could be 

 readily explained. The factor, by which the equihbrium con- 

 stant K is multiplied, is equal to the ratio between the dissocia- 

 tion constants {D^^ and D^^^) of the oxidized and the reduced 

 enzyme-coenzyme compounds. And the redox potential of the 

 free coenzyme is changed by the coupling to enzyme protein 

 according to a simple formula 



Eo (holoenzyme) = E^ (free coenzyme) -\ ^^ In — -^ (a) 



In the case of ADH this means that E'q for the holoenzyme is 70 

 mv. higher than Eq for the free DPN-DPNH system in the 

 physiological pH range. 



The calculated Eq values for the holoenzyme at varied pH 

 indicated the slope dE'jdpH to be -0.030 v. from pH 6.4 to 

 7.8, and —0.060 v. above j&H 7.8. This indicated the existence 

 of an acid group with /?K' = 7.8 in the DPN-ADH. There 

 might be a corresponding group in DPNHADH with a /)K 

 around 10, but convincing experimental data are difficult to 

 obtain at this high alkalinity. The chemical nature of the 

 group with />K' = 7.8 has not yet been established, but as we 

 shall discuss below, it is certainly of decisive importance for the 

 kinetics of the whole enzyme system. 



From a thermodynamic point of view, DPN would appear 

 very unsuitable for oxidizing ethanol. As easily seen from the 

 value of A', lO^^^, the reaction between DPN and ethanol plus 

 catalytic amounts of ADH at pH = 7 would come to equilib- 

 rium when, for example, the ratio DPN:DPNH is = 1, and the 

 ratio ethanol: aldehyde is = 10,000. After the coupling to the 

 protein this last ratio is much more favorable for ethanol oxi- 



295 



