LOCATION OF THE BINDING-SITE FOR THE ALLOSTERIC ACTIVATOR IMP IN THE COOH-TERMINAL DOMAIN OF ESCHERICHIA-COLI CARBAMYL-PHOSPHATE SYNTHETASE

Using UV-irradiation we cross-linked IMP, the allosteric activator of E. coli carbamyl phosphate synthetase (a heterodimer of 117.7 and 41.4 kDa subunits), to the large subunit of the enzyme. As in the native enzyme-IMP complex, the cross-linked complex was resistant to attack by trypsin. Thus, IMP is attached to its normal site and induces the normal conformational changes. Limited digestion of the [H-3]IMP-labeled enzyme with V8 staphylococcal protease or with trypsin in the presence of SDS, and NH2-terminal sequencing, showed that [H-3]IMP is cross-linked to the COOH-terminal 20 kDa domain of the large subunit, downstream of residue 912, supporting the proposal that this domain is specialized in effector binding and regulation. (C) 1994 Academic Press, Inc.