VOL. 12 (1953) CHOLINE ACETYLASE SPECIFICITY 323 



SUMMARY 



The specificity pattern of choline acetylase was tested with partially purified preparations, 

 obtained from head ganglia of Squid, of a specific activity of about 40 to 80 [j,M of ester formed 

 per mg protein per hour. The reaction mixture contained in addition to the enzyme only acyl deriva- 

 tives of CoA and amino ethanols methylated with a varying number of methyl groups as substrates. 



The acetylation of choline was tested as a function of acetyl CoA, of choline and of enzyme 

 concentration. The choline curves showed marked saturation corresponding to a Michaelis-Menten 

 constant of about 2-10-^ M. The acetyl CoA showed less saturation in the range tested; the data 

 correspond to a Michaelis-Menten constant of the order of 5- io~^ M. 



A striking differences was obtained in the acetylation of trimethyl, dimethyl and monomethyl 

 ethanolamine. With the di- and monomethyl compounds the acetylation was only 8 and 2 % re- 

 spectively, compared to that of trimethyl aminoethanol. This strong distinction by the enzyme 

 toward the 3rd methyl group becomes of great biological interest in view of comparably strong 

 effects of the 3rd methyl group towards the other proteins of the acetylcholine system. Loss of 

 the 3rd methyl group leads to nearly complete loss of action upon the receptor protein which de- 

 termines nerve membrane characteristics associated with such phenomena as ion permeabihty and 

 electric potential. In the reactivation process of acetylchohnesterase inhibited by alkyl phosphates 

 similar sharp differences between compounds with 2 and 3 methyl groups are observed in the inter- 

 action with the enzyme. 



In view of the chemically saturated nature of a quaternary group and its tetrahedral structure 

 it is suggested that its unique action may be attributed to an alteration of the protein configuration 

 so as to envelope the molecule. This appears necessary if the protein is to interact simultaneously 

 with the three methyl groups and the chemical functional group of the molecule. Such change in 

 configuration may be necessary for full function of the proteins. 



Among the acyl derivatives of CoA only propionyl CoA has a reactivity comparable to that 

 of acetyl CoA. The enzyme does not mediate the reaction of butyryl CoA with choline, although 

 the latter is bound by the enzyme even stronger than the acetyl CoA. This may have biological 

 significance, since in contrast to propionyl CoA butyryl CoA is an intermediate of fatty acid metabo- 

 lism and a ready formation of this choline ester may have undesirable effects. 



RESUME 



La specificite de la choline acetylase a ete etudiee sur des preparations partiellement purifiees, 

 obtenues a partir de ganglions cephaliques de calmar, et dont I'activite specifique etait d'environ 

 40 a 80 [jlM d'ester forme par mg de proteine et par heure. Le milieu reactionnel renfermait, outre 

 I'enzyme, exclusivement des derives acetyles du CoA et des amino ethanols methyles, portant un 

 nombre variable de groupements methyliques, comme substrats. 



L'acetylation de la choline a ete etudiee en fonction de la concentration en acetyl CoA, en 

 choline et en enzyme. Les courbes de la choline presentent une saturation marquee correspondant 

 a une constante de Michaelis-Menten d'environ 2 • 10-=^ M. L'acetyl CoA presente une saturation 

 moins nette dans le domaine des concentrations employees; les resultats correspondent a une constante 

 de Michaelis-Menten de I'ordre de 5 • lo-^ M. 



Une difference frappante existc entre l'acetylation de la trimethyl-, de la dimethyl- et de la 

 monomethylethanolamine. L'acetylation des composes di- et monomethyles n'est que 8 et 2% 

 respectivement, de celle du trimethylaminoethanol. La forte specificite de I'enzyme vis a vis du 

 3eme groupe methylique presente un grand interet biologique, etant donne les effets comparables 

 du 3eme groupe methylique vis a vis d'autres proteines du systeme de I'acetylcholine. La perte du 

 3eme groupe methylique entraine une perte d'action presque totale sur la proteine recepteur qui 

 determine les proprietes de la membrane de la fibre nerveuse, associees a des phenomenes tels que 

 la permeabilite aux ions et le potentiel electrique. Au cours de la reactivation de I'acetylcholinesterase 

 inhibee par des alkyl phosphates, des differences pareilles entre les substances avec deux et trois 

 groupes methyliques s'observent dans I'interaction avec I'enzyme. 



Etant donne le caractere sature d'un groupe quaternaire et sa structure tetraedrique, on pent 

 supposer que sa seule action pent etre une alteration de la configuration de la proteine telle que cette 

 derniere enveloppe la molecule. Ce phenomene doit etre necessaire, si la proteine doit reagir a la 

 fois avec les trois groupes methyliques et le groupe chimique fonctionnel de la molecule. De telles 

 modifications de configuration semblent necessaires pour que I'activite des proteines soit totale. 



ZUSAMMENFASSUNG 



Die Spezifitat der Cholinacetylase wurde mit teilweise gereinigten, aus den Kopfganglien des 

 Tintenfisches erhaltenen, Praparaten einer spezifischen Aktivitat von 40-80 fxM gebildeten Esters 



References p. 324. 



