Reactions of Glutaminase with 6-Diazo-5-oxonorleucine

In the presence of an excess of the glutamine analog, DON, the enzyme is readily inactivated irreversibly (7). This substance behaves as an active-site-directed alkylating agent in a number of enzymes which utilize glutamine as substrate (3). With the use of 6-I4C-DON, the amount of inhibitor covalently bound to protein can be directly measured and, as shown in Fig. 1, this amount is linearly correlated with the extent of inhibition of catalytic activity. The simplest explanation of this behavior is that irreversible reaction of one molecule of DON with the enzyme inactivates one catalytic site, probably by combination with an essential group at that site. With this assumption the concentration of active sites in an enzyme preparation may be calculated for the 

It may be seen from the proportional region of Fig. 1 that only a small fraction, about 1/70, of the DON added to the enzyme becomes covalently bound. Further analysis of this phenomenon has shown that glutaminase catalyzes the hydrolytic cleavage of the ketone and that the rate of this reaction is 70 times that of the irreversible inactivation of the enzyme. Once alkylation of all reactive sites is complete, the further catalytic hydrolysis of DON is totally blocked. 

After reaction of 6-14C-DON with glutaminase, glutamic acid (1 equivalent) and I4C-methanol (0.75 equivalent) were isolated from the reaction mixture. The initial product formed from the C-6 atom appears to be diazomethane Ha). If relatively high concentrations of a benzoic acid-benzoate buffer are included in the reaction mixture, labeled methyl benzoate is formed in addition to the methanol. Further, the relative 

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