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Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with 6-diazo-5-oxo-L-norleucine resulted in complete loss of its ability to catalyze glutamine-dependent phosphoribosylamine formation and its glutaminase activity, whereas its ability to catalyze ammonia-dependent phosphoribosylamine formation and to hydrolyze phosphoribosylpyrophosphate was increased. The site of reaction with 6-diazo-5-oxo-L-norleucine was the NH2-terminal cysteine residue. The NH2-terminal sequence of the B. subtilis enzyme was homologous with that of the corresponding amidotransferase from Escherichia coli, for which the NH2-terminal cysteine is also essential for glutamine utilization (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536). The fact that the metal-free E. coli amidotransferase contains a glutamine-utilizing structure that is very similar to that found in B. subtilis amidotransferase, which contains an essential [4Fe-4S] center, indicates that the iron-sulfur center probably plays no role in glutamine utilization.