Aromatic N-deacylation by chick-kidney mitochondria

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Abstract
Homogenates of chick kidney rapidly deacylate a variety of acylanilides. Activity is associated with the mitochondria, from which it cannot be released by treatment with several surface-active agents or by repeated freezing and thawing. Some properties of the enzyme have been investigated in mitochondrial suspensions. Deacylation is most rapid in phosphate buffer at pH 7.3 and at 44[degree]. Heat of activation has been calculated as 15,300 cal. Heavy metals inhibit the enzyme strongly. At concentrations as low as 0.2 [mu]M, Hg++ inhibits by about 20%. Inhibition by p-chloromercuribenzoate (50% at 50 [mu] [image]) is partly reversed by reduced glutathione and cysteine. Substitution in the para position of acetanilide generally results in an increase in the relative rate of deacetylation and a decrease in apparent Km. Ortho substitution has a marked effect in the opposite direction; the decrease in rate (which may be 1000-fold) at moderate substrate concentration can be accounted for only partly by decreased affinity of substrate for enzyme. The effect of increasing the N-acyl chain length from formyl to hexanoyl has been tested. Hydrolysis is fastest at acetyl or propipnyl, depending upon the nature of the para substituent. Of all the substrates tested p-acetamidophenylacetic acid has the lowest apparent Km (78 [mu][image]). Chloroacetylated compounds are hydrolyzed most rapidly; p-chloroacetamidoanisole, for instance, is deacylated at a maximal rate corresponding to 6.1 m-moles/g of fresh tissue/hr. Some N-benzoyl derivatives of p-aminobenzoic acid are slowly hydrolyzed. On the basis particularly of distribution, subcellular localization, sensitivity to metal inhibition and the effect on rate of ring substitution and N-acyl chain length the enzyme is seen to have properties very different from those of kynurenine formamidase.