3l6 S. HESTRIN VOL. 4 (1950) 



increase of ester concentration at equilibrium may be expected. The values found for 

 the K of the choline esterifications approximated 0.2 within the limits of the experi- 

 mental error*. The reasonably good constancy of the values for K despite the large 

 variation of the absolute concentration of ester at equilibrium in the investigated pfj 

 range supports the suggestion that undissociated acid rather than the anion enters into 

 the equilibrium of the esterification. 



A value for the A F oi choline ester hydrolysis may be calculated from K with 

 the aid of the relationship 



55-5 



-ZIF = RTln 



K 



whose derivation has been discussed recently by Meyerhof and Green^*. — Zl F calcu- 

 lated in this manner was found to approximate 3200 cals. Although molarities rather 

 than activities are used above to calculate K, it is believed likely that error from this; 

 cause in the value for A F does not exceed 10%**. It is noteworthy that the value for 

 A F oi hydrolysis of two choline esters is of an order similar to the observed in the case 

 of several anionic esters^'*. 



The amount of the acetylcholine at equilibrium is minute in comparison to the 

 concentration of the other participants of the system. However, it seems desirable in 

 view of the great biological potency of acetylcholine to consider the possibility that 

 esterase functions as an agent of acetylcholine synthesis in vivo, supplementing in this 

 respect the role of choline acetylase. It has been demonstrated that acetylcholine 

 esterase in the nerve axon is localized in the neuronal surface membranes^^. The con- 

 centration of esterase substrates and the pn prevailing in the membrane are unknown, 

 but there is reason to believe that H+ and choline+ may be significantly higher at the 

 membrane interface than in the surrounding milieu^^. Specific binding of ester and 

 sudden variation in pf£ at the membrane with resulting shift of equilibrium are con- 

 ceivable. For a local choline concentration of o.oi M and a similar concentration of 

 undissociated acetic acid, the value 0.2 for K leads to an equilibrium acetylcholine 

 concentration of 0.06 micrograms per ml. An ester concentration of this order would 

 be sufficient to produce major biological effects. 



C. FORMATION OF HYDROXAMIC ACIDS 



The ability of proteolytic enzymes to catalyse ester hydrolyses has been demon- 

 strated by Neurath and his coworkers^'. The ability of 0-acyl hydrolases-lipase^^ and 

 esterase^ to form hydroxamic acids by the condensation of fatty acid with hydroxyl- 

 amine is an interesting counterpart to this situation in which a group of hydrolases 

 catalyses both O- and N-acylation. 



The effect of reactant concentrations on the rate of the formation of hydroxamic 

 acid in the presence of the electric tissue esterase is shown by experiments summarized 

 in Fig. 5. Within a wide range of reactant concentration the relation between reaction 

 rate and reactant concentration remains almost linear. Reactant concentrations up to 

 0.75 M or higher failed to saturate the enzyme. Its affinity for acetate, propionate, and 



* Inaccuracy in the measurement of p^ would exert a relatively large effect on the value of K. 

 The computation of K for pn above 6 suffers from an additional inaccuracy because the concentration 

 of ester approached the limit of the ester determination as the pn increased above 6. 



** I am much indebted to Professor O. Meyerhof for the discussion of this question. 



References p. 321. 



