Chemical modification of a catalytic antibody that accelerates insertion of a metal ion into porphyrin: essential amino acid residues for the catalytic activity

Catalytic antibody 2B generated by immunization with N-methyl mesoporphyrin as hapten catalyzes the insertion of a cupric ion into mesoporphyrin. To identify amino acid residues essential for the catalytic activity, we studied effects of various amino acid-reactive reagents on the catalytic activity. The reagents reactive to Arg, Tyr and carboxyl-containing residues inactivated the antibody and mesoporphyrin protected notably the antibody from the inactivation. These results indicated that Arg, Tyr and carboxyl-containing residues are situated in or near the substrate-binding site of the antibody and that some of them would be essential for the catalytic activity. The modified Arg and Tyr residues in the inactivation were quantified in connection with the residual activity. As the result, it was shown that three Arg and one Tyr residues are modified to lead the inactivation. Kinetic analysis indicated that the antibody loses the catalytic activity by modification of one carboxyl-containing residue. In order to find candidates for the modified residues, we performed modeling of the variable domain of the antibody. The model showed that the modified residues are Arg L54, Arg H94, Arg H95, Tyr L91 and Asp H96, and suggested that Arg H95, Tyr L91 and Asp H96 residues would stabilize the transition state of mesoporphyrin in the antibody-mediated reaction.