VOL. 12 (1953) CHOLINE ACETYLASE SPECIFICITY 319 



TABLE I 



Acetylation of choline by choline acetylase as function of enzyme concentration. 

 The reaction mixture contained in addition to the enzyme as indictated the following compo- 

 nents in ^M per ml: Acetyl Co A 5, choline 20, phosphate buffer pH 7,90. Total volume i ml, t = 3i°C. 



The approximate linearity of rate with time and enzyme concentration and the 

 demonstration of enzyme saturation indicates that we have a stable enzyme system 

 which we may reasonably anticipate to be free of complicating and unknown second- 

 ary features. 



The enzyme specificity toward the acetyl acceptor with regard to extent of methyla- 

 tion of the aminoethanol is shown in Table IL The large difference between trime- 

 thyl and dimethyl aminoethanol is of interest in comparison with the results obtained 

 with less purified systems, in which case the trimethyl compound was only twice as good 

 as the dimethyl. In crude preparations no difference was observed between the two 

 compounds^^. However, in those experiments acetyl CoA was not a substrate but only a 

 catalytic intermediate being formed from acetate, ATP and CoA, and mediated by 

 acetylkinase. 



TABLE II 



Effect of the number of methyl groups upon the acetylation of aminoethanols by choline acetylase. 



The reaction mixture contained in addition to the enzyme as indicated below the following com- 

 ponents in jxM per ml: Acetyl CoA 5, aminoethanols 80, phosphate buffer pH 7, 50. Total volume 

 i.o ml, / = 31 °C. 



Methylaminoelhanols acetylated in nMjml 



Trimethyl Dimethyl Monomethyl 



Enzyme (mg) 0.14 1.4 1.4 



Time (min) 



15 0.61 0.37 0.12 



30 1.08 0.76 0.18 



60 1.64 1. 31 0.28 



The increased disparity between the utilization of the two compounds with 

 increased purity indicates that two (or more) enzymes are involved, at least in the crude 

 preparations. We may not conclude, therefore, that the specificity pattern with the 

 present enzyme preparation is necessarily that of a single enzyme but we can say that 

 the major enzyme constituent is at least as discriminating in regard to a third methyl 

 group as is indicated in the Table. Since the concentrations of the amino alcohols were 

 very much higher than the Michaelis-Menten constant for choline the distinction is in 

 the saturation rate and the question of relative binding is not involved. 



The specificity pattern with respect to acyl CoA is shown in Fig. 3. In this case it 

 would appear that both binding and rate factors are involved since the acyl CoA con- 



References p. 324. 



