Protease Activity of 1,10-Phenanthroline−Copper(I). Targeted Scission of the Catalytic Site of Carbonic Anhydrase

• Published in: Biochemistry (Easton) Vol. 37; no. 8; pp. 2096 - 2104
• Main Authors: Gallagher, James, Zelenko, Ottilie, Walts, Avram D, Sigman, David S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 24-02-1998
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Carbonic Anhydrases - chemistry; Cattle; Copper - chemistry; Endopeptidases - chemistry; Humans; Ligands; Models, Chemical; Models, Molecular; Molecular Structure; Peptide Fragments - chemistry; Phenanthrolines - chemistry; Protein Conformation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi971565j

To investigate the mechanism of scission of proteins by the chemical cleaving agent 1,10-phenanthroline−copper, the active sites of human carbonic anydrase I and bovine carbonic anhydrase II have been targeted for cleavage by a tight binding sulfonamide inhibitor tethered to the metal complex. The inhibitor−phenanthroline−copper conjugate binds to the carbonic anhydrases with sub-micromolar K d's and, upon addition of a reducing agent, causes scission specifically within the active site of the enzymes to yield a discrete set of cleavage fragments. N- and C-terminal sequencing and mass spectrometric analysis of several fragments indicate that the C-terminal cleavage fragments have free amino groups at their N termini, thereby allowing facile location of the cut sites through standard Edman degradation. The N-terminal cleavage fragments do not have a free carboxyl group at their C termini. It is proposed that scission occurs by abstraction of H at Cα, followed by oxidation at Cα by the neighboring cupric ion and cleavage of the Cα−C(O) bond to give an N-terminal fragment containing a C-terminal acyl amide, and an unstable C-terminal fragment containing an N-terminal isocyanate group which undergoes hydrolysis to a free amino terminus. Modeling of the inhibitor−phenanthroline−copper conjugate within the active site of human carbonic anhydrase I shows that the sites of cleavage that have been identified are fully consistent with the available structural data.