Active site topology of artificial peroxidase‐like hemoproteins based on antibodies constructed from a specifically designed ortho‐carboxy‐substituted tetraarylporphyrin

Abstract

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)-A,A,A,β-meso-tetrakis(ortho-carboxyphenyl)porphyrin {A,A,A,β-Fe [(o-COOHPh) 4 -porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H 2 O 2 . In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.

New biocatalysts mimicking oxidative hemoproteins: Hemoabzymes

“…Among the seven monoclonal antibodies which recognized MP8, the one that had the best affinity for MP8, IgG1 3A3, bound it with an apparent K d value of 10 À7 M, which was in the range of the apparent K d values already described for IgGemetalloporphyrin complexes [70,95].…”

“…Indeed, two particular classes of antibodies raised against N-substituted [55,61,68] or meso-carboxyaryl-substituted [62,65,70] porphyrins have shown, in the presence of the corresponding iron(III)-porphyrin cofactor, a significant peroxidase activity characterized by efficiencies ranging between 3.7 Â 10 3 and 2.9 Â 10 5 M À1 min. À1 .…”

“…This is probably what occurs in the antibodyemetalloporphyrin complexes. The results of Green et al [56], as well as the results on the study of the binding site of the 13G10 antibody [70,81], show that in those antibodies about two thirds of the porphyrin are included in the binding site leaving, one third of the porphyrin exposed to the solvent. On the contrary, in the case of cytochrome P450s, the substrate enters the active site and binds in a hydrophobic pocket, over the plane of the porphyrin close to the iron(V)-oxo species.…”

“…Finally, a comparison of the K d s for the four atropoisomers of ToCPPH 2 (Fig. 2B) [70] showed that mainly three of the ortho-carboxylate substituents of the phenyl rings, in a,a,b positions, were recognized by amino acids of the antibody protein. This led us to propose a possible binding site topology in which roughly two thirds of the porphyrin macrocycle could be inserted in the binding pocket, with two adjacent a,a-carboxylates being more specifically bound to the protein.…”

“…UVevisible spectroscopy studies, associated with the determination by competitive ELISA of the apparent dissociation constants (K d ) for various porphyrins have allowed us to propose a possible binding site topology of antibodies 13G10 and 14H7 [70]. Both studies suggested first that the central metal atom of the hapten was not recognized by the antibody protein since a,a,a,b-Fe-and -Mn-ToCPP, as well as the free-base a,a,a,b-ToCPPH 2 , all bound to 13G10 with similar K d s [70]. This showed that no amino acid residue was induced in the antibody protein that was able to coordinate the iron atom of the hapten.…”