Rat liver ω-amidase catalyzes hydrolysis and acyl-transfer reactions with the amide and monoalkyl esters of α-ketoglutarate, glutarate, and succinate. The specificity of the enzyme has been extended by demonstrating the reactivity of a number of para-substituted phenyl glutarates. p-Methoxy, p-chloro, p-methyl, and the unsubstituted phenyl ester of glutarate are hydrolyzed at about one-half the rate of the methyl ester, and show little dependence on the pK of the leaving group. Hydroxaminolysis of glutaramate and methylglutarate leads to a twofold increase in the total rate of substrate utilization (hydrolysis plus hydroxaminolysis), with a nonstoichiometric decrease in hydrolysis at lower hydroxylamine concentrations. On the other hand, hydroxylamine has no effect on the rate of p-cresol release from p-methylphenyl glutarate, and the rate of hydrolysis is decreased proportionally to the increase in hydroxaminolysis. The partitioning of the acyl group derived from glutaramate, methyl glutarate, and p-methylphenyl glutarate between water and hydroxylamine is identical for the three substrates. Nucleophiles such as hydroxylamine and methanol show uncompetitive activation for the total rate of glutaramate disappearance, and for a particular acyl donor Vmax for acyl transfer is independent of the added nucleophile. These results are interpreted in terms of a two-step mechanism involving an acyl-enzyme intermediate.
|Number of pages||6|
|State||Published - Jun 1 1972|
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