3i8 



S. HESTRIN 



VOL. 4 (1950) 



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120 



180 



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Minufes 



hydroxylamine, and some enzymatic catalysis 

 of this reaction. Substitution of an a-amino 

 group into acetate or propionate caused com- 

 plete loss of their ability to condense with 

 hydroxylamine in the presence of the enzyme. 

 The ability of the esterase to effect hydro- 

 lysis of acethydroxamic acid was examined at a 

 substrate concentration of 3 /^M/ml in phosphate 

 buffer at pn 74. Even with a great concentration 

 of enzyme no hydrolysis of acethydroxamic 

 acid was found, although acetylcholine added to 

 the same reaction mixture was hydrolysed 

 rap'dly. An acyl transfer reaction between acet- 

 hydroxamic acid and chohne with resultant 

 intermediary formation of hydrotysable acetyl- 

 choline could be excluded, since addition of 

 choline to the same reaction mixture failed to 

 evoke a disappearance of acethydroxamic ac'd. 

 The Ph dependence of hydroxylamine 

 acylation by electric tissue esterase is illus- 

 trated by the experiment g'ven in Table V. 

 The reaction between acetate and hydroxyl- 

 amine showed a peak in a range near pfj 6.3. 

 The Ph function of hydroxylamine acylation 

 by the esterase is thus very different from the pn function of acetylchohne h5^drolysis 

 by the enzyme. The finding that the pn dependence of hyd'-oxylamine acylation and 

 choline ester hydrolysis are quite different is consistent with an assumption, discussed 

 later, concerning the mechanism of these two reactions. 



TABLE V 



FORMATION OF HYDROXAMIC ACID IN PRESENCE AND ABSENCE OF CHOLINE AT DIFFERENT pjj 



Reaction mixtures are 0.5 M as to acetate and i.o M as to hydroxylamine in 0.9 M solutiDn of sodium 

 chloride at pn specified with or without addition of 0.5 M choline chloride. In absence of choline 

 addition, an equivalent amount of sodium chloride was added. The pn was determined in aliquots 

 with a glass electrode after four-fold dilution with water. Temperature 37° C. The formation of 

 hydroxamic acid in absence of enzyme was «negligible at pH 6.3 and 5.3 and none was detected at 

 Ph 7 and higher. The reaction time was 4 hours. 



Fig. 6. Formation of hydroxamic acid as 

 a function of esterase concentration. Re- 

 action mixtures were i.o M as to hydroxyl- 

 amine and sodium acetate in 1.0 molar 

 sodium chloride, pn 6.8. 37° C. Curves 

 I to 3 correspond to relative enzyme 

 concentrations 10, 6, and 3. In the inset 

 the relative reaction rate is plotted on 

 the ordinate and the relative enzyme 

 concentration on the abscissa. 



In the presence of choline, the rate of the acetylation of hydroxylamine by esterase 

 References p. 321. 



