A MOLECULAR WEDGE FOR TRIGGERING THE AMIDOTRANSFERASE ACTIVITY OF CARBAMOYL-PHOSPHATE SYNTHETASE

The reactive cysteine residue within the small subunit of Escherichia coil carbamoyl phosphate synthetase has been identified using the tech nique of site-directed mutagenesis. Three cysteine residues have previ ously 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 subuni t. In the present investigation, Cys-248 of the small subunit has been identified as the residue that reacts with NEM in the presence of MgA TP and bicarbonate. Three cysteine residues of the small subunit at po sitions 131, 214, and 248 were individually mutated to serine residues . These site-specific changes, in addition to N-ethylmaleimide-labelin g studies, demonstrated that Cys-248 is the amino acid that reacts wit h N-ethylmaleimide. Substitution of Cys-248 of the small subunit with larger residues (Asp, Phe, Arg, and Trp) was conducted in order to mor e closely mimic the observed properties of the NEM-labeled enzyme. The partial glutaminase activity of the C248D mutant increased 40-fold re lative to the wild-type enzyme, while the formation of carbamoyl phosp hate using glutamine as a nitrogen source was completely abolished. Si milar, but less dramatic, effects were observed for the other mutants, C248S, C248R, C248F, and C248W. There was good correlation between th e extent of enhancement of the partial glutaminase activity and an unc oupling of the phosphorylation reactions that occur on the large subun it. These results have been interpreted to suggest that replacement of Cys-248 with bulkier residues induces the conformational changes in t he small subunit that are thought to occur upon binding of MgATP and b icarbonate to the large subunit. The bulkier substituents serve as a m olecular wedge for prevention of the native conformation and as a trig ger for the enhancement of the hydrolysis of glutamine.