VOL. 4 (1950) 



ENZYMATIC CONDENSATIONS WITH NHjOH 



305 



is considerably smaller in the case of hydroxamic acid formation. The dependence of 

 lipatic hydroxamic acid condensation on higher concentrations of hydroxylamine sug- 

 gested a near equilibrium situation. Therefore, the influence of the concentration of 



TABLE V 



COMPARISON OF HYDROXAMIC ACID FORMATION AND TRIBUTYRIN 

 HYDROLYSIS WITH VARIOUS HOG LIVER FRACTIONS 



For the hydroxamic acid determination o.i of the original fraction was used in a total volume of 

 0.5 ml, hydroxylamine 0.6 M, and octanoate 0.02 M, and incubated for 60 minutes at 37°. 

 Tributyrin hydroh'sis was measured mano metrically with the manometer containing the fraction 

 in appropriate dilutions, Li:i/i2; L2:i/i2; L3:none; L4:i/2o. The vessels contained o.i ml of 

 the diluted fraction, 0.6 ml of 0.1 M Na bicarbonate and 0.05 tributjTin was dipped in from the side 

 arm. The gas room contained 5% COg in N^. To make the two series comparable the values recorded 

 in the table for the manometric experiment were obtained by multiplication with the respective 

 dilution factors. 



the other reaction partner, the carboxyl ion, 

 was likewise tested. In Fig. 2, two concen- 

 tration levels, 0.02 and 0.2 molar are com- 

 pared. The expected increase with carboxylate 

 concentration is most evident at intermediate 

 chain lengths. With longer chain lengths the 

 often observed inhibition by free long-chain 

 fatty acid overlaps. This also explains the 

 change of the chain length optimum toward 

 shorter chains at higher concentration, due 

 to increasing hydrolysis of the salt at higher 

 concentration levels. It is of special interest 

 that the acetate ion starts to show appreciable 

 activity at the 0.2 molar level. 



In Fig. 3 the time curve of the reaction 

 is traced. It appears that, with the reactants 

 present in excess, the condensation occurs 

 practically proportionally with time, indi- 

 cating, as would be expected, an enzymatic 

 reaction of the zero order. 



Although in the experiment with carb- 

 oxylate ion an intermediate formation of 

 an ester was seemingly excluded, it appeared 

 nevertheless of interest to explore the pos- 

 sibility of rapid enzymatic conversion of ester 

 into hydroxamate. For this purpose, the en- 

 zyme was incubated with equivalent amounts of tributyrin and butyrate. As shown 

 References p. 309. 



4 5 



Fig. 2. Comparison of hydroxamic acid 

 formation with 0.02 and 0.2 M octanoate, 

 0.6M hydroxylamine, and 0.1 ml enzyme 

 solution in 0.5 ml total volume, 60 minutes 

 incubabation at 37°. 



