The reactive cysteine residue within the small subunit of Escherichia coli carbamoyl phosphate synthetase has been identified using the technique of site-directed mutagenesis. Three cysteine residues have previously been found to react with N-ethylmaleimide (NEM) under controlled reaction conditions. Two of these cysteine residues are found on the large subunit, while the third cysteine is located on the small subunit. In the present investigation, Cys-248 of the small subunit has been identified as the residue that reacts with NEM in the presence of MgATP and bicarbonate. Three cysteine residues of the small subunit at positions 131, 214, and 248 were individually mutated to serine residues. These site-specific changes, in addition to N-ethylmaleimide-labeling studies, demonstrated that Cys-248 is the amino acid that reacts with N-ethylmaleimide. Substitution of Cys-248 of the small subunit with larger residues (Asp, Phe, Arg, and Trp) was conducted in order to more closely mimic the observed properties of the NEM-labeled enzyme. The partial glutaminase activity of the C248D mutant increased 40-fold relative to the wild-type enzyme, while the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Similar, but less dramatic, effects were observed for the other mutants, C248S, C248R, C248F, and C248W. There was good correlation between the extent of enhancement of the partial glutaminase activity and an uncoupling of the phosphorylation reactions that occur on the large subunit.(ABSTRACT TRUNCATED AT 250 WORDS)