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Asparagine Utilization in Escherichia coli

^a Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92664

ABSTRACT

Asparagine-requiring auxotrophs of Escherichia coli K-12 that have an active cytoplasmic asparaginase do not conserve asparagine supplements for use in protein synthesis. Asparagine molecules entering the cell in excess of the pool required for use of this amino acid in protein synthesis are rapidly degraded rather than accumulated. Supplements are conserved when asparagine degradation is inhibited by the asparagine analogue 5-diazo-4-oxo-L-norvaline (DONV) or mutation to cytoplasmic asparaginase deficiency. A strain deficient in cytoplasmic asparaginase required approximately 260 µmol of asparagine for the synthesis of 1 g of cellular protein. The cytoplasmic asparaginase (asparaginase I) is required for growth of cells when asparagine is the nitrogen source. This enzyme has an apparent K_m for L-asparagine of 3.5 mM, and asparaginase activity is competitively inhibited by DONV with an apparent K_i of 2 mM. The analogue provides a time-dependent, irreversible inhibition of cytoplasmic asparaginase activity in the absence of asparagine.

¹ Present address: Department of Biochemistry, University of California, Riverside, California 92502.