VOL. 4 (1950) 



ACYLATIONS BY ACETYLCHOLINE ESTERASE II 



317 



hydroxylamine may be concluded, therefore, to 

 be of a much lower order than the afifinity of 

 the enzyme for acetylcholine. This conclusion 

 has been further supported by the demonstra- 

 tion that neither acetate nor hydroxylamine 

 significantly affect the rate of acetylcholine 

 hydrolysis by the esterase. The substrate con- 

 centration-activity relationship observed in 

 hydroxylamine acylation resembles that of 

 neutral ester hydrolysis by the enzyme^' ^. 



The rate of reaction of acetate with hydr- 

 oxylamine in the presence of esterase is very 

 small as compared to the rate of hydrolysis of 

 acetylcholine by a similar concentration of the 

 enzyme, the relative magnitude of the rates 

 being in the proportion of one to one or two 

 thousand. The rate of hydroxamic acid forma- 

 tion, like the hydrolysis of acetylcholine, varied 

 in a direct manner with the esterase concentra- 

 tion (see Fig. 6). 



The specificity of electric tissue esterase in 

 regard to the fatty acids which it can cause to 

 condense with hydroxylamine is rather sharply 

 defined (see Table IV). As in choline ester 

 hydrolysis^, a maximum is observed with 

 acetic acid. A lower rate is found with propionic 

 acid. The enzyme-catalyzed reaction observed 

 with butyric acid was almost negligible. The 

 findings with formic acid reveal a relatively 

 large spontaneous reaction between formate and 



Fig. 5. Formation of hydroxamic acid as 

 a function of reactant concentration. The 

 reaction mixtures are 0.5 M as to sodium 

 acetate and i.o M as to sodium chloride. 

 Ph 6.8. 37° C. n , ▼ , X , O , — correspond 

 to mixtures with o.i, 0.2, 0.5, and i.o M 

 hydroxylamine respectively. Curves i to 3 

 of the inset are not mutually comparable 

 since they were obtained with different 

 batches of the enzyme. Relative reaction 

 rates are plotted on the ordinates and 

 reactant concentrations in molarity on the 

 abscissae. Curve i summarizes the detail 

 of the main part of the figure showing the 

 effect of variation of hydroxylamine con- 

 centration. Curves 2 and 3 show the effect 

 of variation of acetate and propionate con- 

 centration respectively in the presence of 

 1.0 M hydroxylamine. 



TABLE IV 



SUBSTRATE SPECIFICITY OF ELECTRIC TISSUE ESTERASE IN FORMATION OF HYDROX.\MIC ACID 



The reaction mixtures are 1.0 M as to hydroxylamine and 0.75 M as to the sodium salt of the fatty 



acid in 0.9 M solution of sodium chloride at pn 6.2-6.4. Temperature 37° C. Propionhj'droxamic, 



butyrhydroxamic, and acethydroxamic acid yield equivalent amounts of colour per mole with ferric 



chloride. The amount of the formhydroxamic acid is calculated on the same basis. 



References p. 321. 



