HUGO THEORELL 



both the "on" velocities, ki and k^, to about half. This was not 

 unexpected, since the same kind of effect occurs with FMN + 

 apoprotein in the alkaline />H range ; nor was the increase in k^ 

 unexpected for the same reason. But the decrease in ^3 by 50% 

 was indeed unexpected. The mechanism leading to this result 

 is hard to understand, but its consequences are worth consider- 

 ing. 



First of all, whether chloride will increase or decrease the 

 reaction velocity is found to depend on the concentration of the 

 reaction participants. In a system where DPN and alcohol are 

 both in excess, chloride stimulates the reaction, because k2 is 

 then rate-limiting, and kz is increased by chloride. If now the 

 concentration of DPN is decreased, so that kf, begins to be rate- 

 limiting, chloride inhibits because of its decreasing effect on kf,. 

 Thus the Lineweaver-Burke plots for varied DPN cross one 

 another at a point, where chloride apparently has no effect at all. 



Suppose we would determine the dissociation constant 

 (DqJ of DPNADH by equilibrium measurements with and 

 without chloride. Practically the same value would come out 

 in both cases, because k^ and k^ are both reduced to half. There 

 is, however, a kinetic difference: in chloride it takes a longer 

 time for DPN and ADH to reach equilibrium. 



The opposite effect of chloride on k^ and k^ leads to a change 

 in D^JD^^^. This means that the oxidation-reduction potential 

 of the enzyme complex will be lowered from —0.204 to —0.219 

 V. if 0.15 M chloride is added to a solution of DPN ADH + 

 DPNHADH. 



Formate, 0.015 M, is found to affect only two of the six 

 constants, kz and k^. This means that formate reacts only with 

 DPN -ADH, but not with DPN or ADH before the complex is 

 formed, since k^ is not changed. Formate may enter into the 

 binding site for ethanol in DPNADH, since it decreased k^ 

 competitively. The formate then stabilizes the linkage of DPN 

 to ADH (decreased Ats) . 



There thus seems to be a strict analogy between the effect 

 of formate and the effect of hydroxylamine, as studied by Kaplan 



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