Evolution of antibody catalysis

Antibody 7G12 was raised against N-methylmesoporphyrin (NMP) and catalyzes the metallation of mesoporphyrin (MP) by Zn2+ with rates comparable to that of the enzyme ferrochelatase. To establish the mechanism of the porphyrin metallation reaction catalyzed by antibody 7G12, we solved the x-ray crystal structure of a catalytically competent Michaelis complex formed between the Fab fragment of antibody 7G12 and its substrate MP. The crystal structure clearly shows the Fab bound MP molecule is distorted toward the transition state conformation for metal insertion and provides definitive structural evidence for the strain theory proposed by Haldane more than seventy years ago.; We are also interested in the immunological evolution of catalytic antibodies. The generation of antibodies by the immune system through V-(D)-J recombination and somatic hypermutation is similar in many aspects to the combinatorial and mutational processes that occur during the evolution of enzymes in nature. As a prototypical example of enzyme evolution, the affinity-maturation processes of catalytic antibodies 7G12 and 28B4 were studied by solving the x-ray crystal structures of the unliganded Fab fragments of the germline antibodies, the germline Fab-hapten complexes and the unliganded Fab of the affinity-matured antibodies. A comparison of these structures with the previously solved crystal structures of the affinity-matured Fab-hapten complexes of antibodies 7G12 and 28B4 reveals that the germline antibody combining sites undergo significant conformational changes upon hapten binding to achieve structural complementarity with their haptens. In contrast, the affinity-matured antibodies show minimal changes upon the binding of haptens, consistent with a "lock and key" type of binding. Thus a greater degree of conformational flexibility exists in the active sites of the germline antibodies, which may play a major role in expanding the binding potential of the germline repertoire. However during affinity maturation, somatic mutations remove the structural flexibility in the germline antibodies and create rigid, preorganized antibody combining sites well adapted for hapten binding. The studies on antibody 7G12 also reveal the mechanism by which the immune system evolves binding energy to catalyze the porphyrin metallation reaction.; The complex of 7G12 Fab and mesoheme was also found to have peroxidase activity, though 104 times lower than that of the enzyme peroxidase. To improve the peroxidase activity of the 7G12 Fab-mesoheme complex, we developed a novel strategy to select 7G12 Fab mutants with enhanced peroxidase activity from a large library of phage displayed mutants based directly on catalytic activity. This selection scheme depends on the ability of biotin tyramine conjugate to covalently crosslink with peroxidase active antibody upon oxidation of the tyramine moiety. The validity of this strategy has been demonstrated in a model selection and some mutants of 7G12 Fab with more than a ten fold increase in peroxidase activity have been identified.