SUBSTRATE INHIBITION 



127 



trations. This salt effect was investigated more thoroughly by Myers (1952 c) 

 who confirmed the earlier results and determined the effects of ionic strength 

 on the various constants, from which it is evident that increasing ionic 

 strength decreases the affinity of the enzyme for acetylcholine but enhances 

 the hydrolytic activity. That this effect is not entirely a nonspecific salt 

 effect is indicated by the fact that the doubly-charged Ca++ ion exerts a 

 stronger action than Na+, since 5 mM CaCla altered the rate-pS curve 

 comparably to 200 mM NaCl. The effects of NaCl concentration on the 

 reaction constants, based on Eq. 4-7, are shown in the tabulation below. 



In this formulation, /i , is the constant for the active ES complex and aKg 

 for the relatively inactive ESg complex (designated K^ and K^ by Myers). 

 The interpretation of aKg as the dissociation constant for the second or 

 inhibiting substrate molecule is probably doubtful, since actually in the 

 ES2 complex there are two different dissociation constants and an in- 

 teraction factor. In the more complete formulation 4-1, what is here desig- 

 nated as a would actually be a^y. If l3K^ refers to the anionic site and 

 yKg to the esteratic site, it might be expected that /? would not differ 

 greatly from unity, inasmuch as the major binding energy of the substrate 

 in the active complex derives from interaction at this site, and that y 

 would be reasonably large. It is difficult to estimate a but it should not be 

 too large since the positive charges on the simultaneously bound molecules 

 are maximally separated and from inhibitor studies the two sites would 

 appear to be well separated. It is thus possible that a value of «/?/ = 200 

 (in the absence of added NaCl) is mainly due to a high value of y. The in- 

 crease in Kg with NaCl concentration would be expected on the basis of a 

 reduced interaction with the anionic site due to increasing ionic strength 

 or competition for the anionic site by Na+. It is difficult to interpret phy- 

 sically the changes in a or a/3y with NaCl concentration because in addi- 

 tion to the possible changes in the dissociation constants themselves, it 

 it is likely that the acidic dissociation of groups at both sites on the enzyme 

 is changed. If the above interpretation is correct, it would appear that the 

 binding to the esteratic site is augmented by high NaCl concentration. In 



