L-asparaginase II of Escherichia coli. Studies on the enzymatic mechanism of action.

The enzymatic mechanism of asparaginase action has been explored using the acyl acceptor, hydroxylamine. Asparaginase catalyzed the synthesis of the hydroxamate from asparagine and more slowly from aspartic acid. P-Aspartohydroxamate was also a substrate for asparaginase. These reactions have rates which are linearly dependent on the concentration of the enzyme and can therefore be used as convenient calorimetric assays. The activity which catalyzed these reactions migrated precisely with asparaginase II during isoelectric focusing with an isoelectric point of 4.9. The reactions with hydroxylamine suggest a mechanism which involves a ,B-aspartyl enzyme intermediate. Further support for this mechanism has been obtained from exchange studies which showed that asparaginase catalyzed the incorporation of oxygen from no-labeled water into aspartic acid. Useful kinetic analysis of the hydrolysis of asparagme cannot be carried out because the concentration of water cannot be varied as a kinetic parameter. The hydroxylaminolysis reactions, however, allowed demonstration of a pingpong mechanism consistent with a reaction pathway involving an acyl enzyme intermediate.