Isolation and sequencing of an active-site peptide from angiotensin I-converting enzyme.

A glutamic acid residue at the active-site of bovine lung angiotensin I-converting enzyme was esterified with p-[N,N-bis-(chloroethyl)amino]phenylbutyryl-L-[U-14]-Proline (chlorambucyl-L-[U-14C]-L-Proline), an affinity label for this enzyme. The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase HPLC contained the bound radiolabel. This active-site peptide (Mr approximately 16,000) was digested with trypsin, and the labeled peptide (T-2) was further degraded with thermolysin. The enzyme digest peptides were also resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained after thermolysin digestion (Th-1, Mr 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu. The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of PTH-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]-Proline which confirms our earlier findings. The sequence that we determined is homologous in five residues with the corresponding sequences of carboxypeptidase A and B, two other mammalian zinc-proteases. There is little sequence homology with thermolysin, a bacterial zinc-protease that also contains an essential active-site glutamic acid residue.