Key residue responsible for catalytic activities in the antibodies elicited against N-methyl mesoporphyrin

Five catalytic and nine non-catalytic antibodies for insertion of a metal ion into porphyrin were generated by immunization with N-methyl mesoporphyrin (N-MMP) as hapten, which was designed to mimic the distortion of porphyrin toward a transition-state geometry in the reaction. In order to determine the features responsible for the catalytic activity, we characterized the properties of the catalytic and non-catalytic antibodies. The catalytic antibodies did not have higher affinity to N-MMP than the non-catalytic ones. All the antibodies, except one non-catalytic antibody, combined with ferric N-methyl mesoporphyrin (N-MMP-Fe) to form the respective antibody·N-MMP-Fe complex. The binding affinity of cyanide to ferric iron in the complexes agreed with that of free N-MMP-Fe, indicating that the protruding side of N-MMP-Fe in the complexes is exposed to solvents. All the complexes of the catalytic antibodies had a peroxidase-like activity, whereas those of the non-catalytic ones did not. This suggests that the metalation activity associates with the peroxidase-like one, so that there is a common residue acting as catalyst for both reactions. The amino acid sequence alignment shows that the catalytic antibodies contain a homologous heavy chain sequence in the third complementarity-determining region. Based on the results, the possibility that Asp(H96) in the region is the key residue responsible for the metalation and peroxidase-like activities is discussed.