GLUTAMINASE ACTIVITY OF L-ASPARAGINE AMIDOHYDROLASE

Relatively high concentrations of 6-diazo-5-oxo-norleucine (DON) and azaserine, potent specific inhibitors of many enzymes using glutamine as substrate, do not have an appreciable effect on the activity of Escherechia coli L-asparagine amido-hydrolase (EC 3.5.1.1) when either asparagine or glutamine is used as substrate. Glutamine acts as a competitive inhibitor when asparagine hydrolysis is measured, and asparagine inhibits glutamine hydrolysis. These observations, together with the fact that no one has been able to separate these two activities of the E. coli enzyme, point strongly to the presence of a single enzymatic site. However, the two activities have different pH dependencies. The glutaminase activity shows a 6-fold increase from pH 5 to pH 8·7; the asparaginase activity is almost constant over this pH range. The measured Michaelis constants and maximum velocities for the glutaminase and asparaginase activities at pH 8·7 are as follows KmG = 1·3 × 10-2M; KwA = 1·3 × 10-3M;VmaxG = 0·080 μmoles pei min per i.u. of asparaginase activity; VmaxA = 1·1 μmoles per min per i.u.The inhibition dissociation constant for glutamine acting as inhibitor of asparaginase in 1·2 × 10-2. © 1969.