Photoaffinity labeling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor

UMP is a highly specific reagent for photoaffinity labeling of the allosteric inhibitor site of carbamyl phosphate synthetase (CPS) from Escherichia coli and has been found to be photoincorporated in the COOH-terminal domain of the large subunit [Rubio ed nl. (1991) Biochemistry 30, 1068-1075]. In the present work we identify lysine 992 as the residue that is covalently attached to UMP. This identification is based on two lines of evidence. First, [C-14]UMP is found to be incorporated between residues 939 and 1006, as shown by peptide mapping and by mass estimates of [C-14]UMP-peptides generated by chemical and enzymatic cleavage of CPS. Secondly, we have purified two radioactive peptides derived exclusively from those enzyme molecules (similar to 5% of the total enzyme) that had incorporated [C-14]-UMP. Edman analyses show the sequences of the labeled peptides (989)LVNXVHEGRPHIQD and (989)-LVNXVHE to be overlapping. Since neither a phenylthiohydantoin (Pth) derivative (in cycle 4) nor any radioactivity is released from the membrane during sequencing, we can conclude that Lys992 and [C-14]-UMP form a covalent adduct that remains bound to the membrane. Formation of this adduct agrees with all of the evidence and with the finding that UMP labeling prevents trypsin cleavage at Lys992. Lysine 992 is invariant in those CPSs that are inhibited by UMP, and is located 30 residues upstream of the site whose phosphorylation in hamster CAD reduces inhibition of CAD by UTP. Multiple sequence alignment of the residues surrounding Lys992 of the E. coli enzyme and the corresponding residues of the yeast and animal enzymes supports the existence of a uridine nucleotide binding fold in this region of the protein. We conclude that sequence changes in the binding fold provide a structural basis for the different regulatory properties found among CPSs I, II, and III.