HUGO THEORELL 



along monovalent dissociation curves of pK. 6.3-6.4. The 

 "off" velocities for these coenzyme-enzyme complexes are thus 

 in contradistinction to O.Y.E. maximal around neutrality. 

 This arrangement is obviously favorable for an enzyme system 

 with a mobile coenzyme. 



As already mentioned above the independent equilibrium 

 data for D^JD^^^ gave a calculated curve for the redox potential 

 of the holoenzyme with an increase in dE'o/dpH at pH 7.8 from 

 — 0.030 to —0.060 v., indicating the presence of an acid group 

 in DPNADH that was absent, or at least had a much higher 

 pK', in DPNH ADH. 



Why is kz so nicely /?H-dependent in the alkaline range, 

 whereas k2 is not? This is very probably connected with the 

 fact that the pyridine ring in DPN has a positive charge in 

 contradistinction to DPNH. If now a group in the protein, 

 H+X~ — R, is close to the pyridine and in this position has a 

 pK. of 7.8, this would result in an extra stabilization (low ^3) 

 of the DPN-ADH linkage in alkaline solution by the ion at- 

 traction in 



Py+X-R 



When X~R adds a proton this stabilization would disappear 

 along a dissociation curve, as is indeed observed. 



The proximity of Py+ to X~R would result in a lowering 

 of the /?K '-value for X~R. In free ADH this group may 

 very well have a/?K' value of 9 or 10, and could thus be a — SH or 

 tyrosyl-OH group. The low value for ^2 indicates that the 

 DPNH-ADH linkage is stabilized by some extra bond in ad- 

 dition to those which must be fairly equal for both the oxidized 

 and the reduced compounds (e.g., pyrophosphate to — NH3). 

 This is made certain by the fact that the 340 mju band of DPNH 

 moves to 325 m/i on coupling to ADH. And this band shift is 

 reversed by low concentrations of j&-chloromercuribenzoate, 

 which completely inhibits the activity of ADH. There is thus 

 good reason to believe that a sulfhydryl group in the protein 

 interacts with the pyridine ring. How this occurs in the case of 



300 



