In vitro abzyme evolution to optimize antibody recognition for catalysis

Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreas ing the affinity for the ground state. To evolve abzymes toward higher cata lytic activity, we have reconstructed an enzyme-evolutionary process in vit ro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to th e ground state. The library format successfully afforded evolved variants w ith 6- to 20-fold increases in activity (k(cat)) as compared with 6D9. Stru ctural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermuta tion in the immune response.