title: Protease activity of 1,10-phenanthroline-copper(I). Targeted scission of the catalytic site of carbonic anhydrase.
abstract: 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 Kd'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 Calpha, followed by oxidation at Calpha by the neighboring cupric ion and cleavage of the Calpha-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.
authors.affiliation: Department of Biological Chemistry, School of Medicine, Molecular Biology Institute, University of California, Los Angeles, California 90095-1570, USA.
authors.forename: J
authors.initials: J
authors.lastname: Gallagher
authors.forename: O
authors.initials: O
authors.lastname: Zelenko
authors.forename: A D
authors.initials: AD
authors.lastname: Walts
authors.forename: D S
authors.initials: DS
authors.lastname: Sigman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D003300:Copper / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry; D010618:Phenanthrolines / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D008024:Ligands; D010446:Peptide Fragments; D010618:Phenanthrolines; C014741:bis(1,10-phenanthroline)copper(2+) ion; D003300:Copper; D010450:Endopeptidases; D002256:Carbonic Anhydrases