VOL. 4 (1950) 



ACYLATIONS BY ACETYLCHOLINE ESTERASE II 



319 





acting at pn 7.1 was reduced markedly (Fig. 7). The _ '^ 

 effect of Ph on the choline inhibition is illustrated g- 

 by the experiment shown in Table V. As in the .^ 

 case of acetylcholine hydrolysis, the lowering of p^ ° 

 reduced the inhibitory effect of choline. At pn 5.3, I 

 a reg'on in wh'ch the enzyme activity was rather | 

 low but still measurable, an activating effect by ^ 

 choline on hydroxamic acid formation was observed. 

 The inhibitory effect of choline can be ascribed to 

 its ability to combine with the enzyme at an active ^ 

 site^". An explanation of activation by choline may 

 be found in the fact that at acid p^ the concentra- 

 tion of acetylcholine in the system is increased. 

 It has been shown" that acetylcholine acetylates 

 hydroxylamine rap'dly at alkaline pn and slowly 

 at acid pn. the rate being dependent on the concen- 

 tration of the acetylcholine at constant hydroxyl- 

 amine concentration. z-\t pn, 7 the concentration 

 of acetylcholine in the acetate-hydroxylamine- 

 choline-system is neglig'ble. The ability of choline 

 to serve as an acetyl carrier at this pn must therefore 

 become very small. 



The inh'bito^y effect of choline on hydroxyl- 

 amine acylation and the finding^ that incubation of 

 the enzyme with specific inhibitors — prost'gmine 

 and tetraethylpyrophosphate — abolishes the abi- 

 lity to catalyse hydT'oxamic acid formation support the view that the same enzyme ,and 

 possibly the same p-osthetic group, effects both acetylcholine hydrolysis and hydrox- 

 amic acid formation. But the reaction of hydrolysis of ac^ tylcholine is reversible, while 

 that of hydroxylamine acylation appea^^s to be irreversible. Choline shows a fairly 

 marked affinity for the enzyme, whereas hydroxylamine shows little or no affinity. The 

 possibility has therefore to be considered that the role of esterase in hydroxylamine 

 acylation is confined to the activation of the carboxylic acid reactant, and that a 

 terminal reaction between activated carboxylic acid and hydroxylamine is spontaneous 

 and irreversible. In the case of choline acylation it is assumed that the esterase may 

 activate the two reactants. 



The writer is deeply indebted to Professor D. Nachmansohx for encouragement 

 and for many suggestions. Thanks are expressed to Mrs Emily Feld Hedal and Miss 

 Louise d'Alessio for their assistance in the performance of the experiments. 



'20 Ao 60 60 100 



Minutes 

 Fig. 7. Effect of choline on formation 

 of hydroxamic acid. The reaction mix- 

 tures are i.o M as to hydroxylamine 

 and 0.75 M as to sodium acetate in 

 0.9 M solution of sodium chloride at 

 Pjj 7.1. Temperature 37° C. O, mixture 

 without choline; X, mixture with 0.9M 

 choline chloride. The reaction in ab- 

 sence of enzyme is negligible in both 

 cases. The inhibitory effect of choline 

 was unaffected by the choline concen- 

 tration in the range of o.i to 0.9 M. 



SUMMARY 



I. Some general properties of ester hydrolysis and synthesis by the purified acetylcholine 

 esterase of the electric tissue of Electrophorus electricus have been investigated with the aid of a 

 simple colorimetric technique for the determination of an ester in the presence of its hydrolysis 

 products. 



References p. 321. 



