Catalytic Antibodies

We are applying a combined theoretical and experimental approach to investigate antibody structure, antigen binding, catalytic mechanism, and the design of metal-binding sites in the catalytic antibody 43C9. 43C9 is unique in its ability to catalyze amide as well as ester bonds. We constructed a model of the variable region (the antigen-binding domain) of 43C9 based on our database of superimposed crystallographic antibody structures. From the model, we predicted two residues to be critical for catalysis: Arg 96 may stabilize the negatively charged transition states and His 91 may be the nitrogen nucleophile that forms the observed acyl-antibody covalent intermediate during the catalytic reaction. These hypotheses have been verified by mutagenesis. In addition, we have designed and constructed several metal-binding sites into the antigen-binding region of 43C9 based on motifs from Zn(II)- and Cu(II)-binding enzymes. One metal-binding site, based on the structure of carbonic anhydrase, shows >100-fold selectivity for Zn(II) over other metals, including Cu(II), Co(II), Cd(II), Ni(II), and Fe(II). Another designed site show a selectivity of Cu(II) over Zn(II). Recently, we determined crystallographic structures of 43C9, both free and with bound p-nitrophenol, a product of esterolysis. Comparison with our previously constructed model has suggested improvements Crystallographic studies are underway to test the computational model of the wild-type 43C9 and to determine the geometry of the designed metal-binding binding sites in the mutants. Thus, computer modeling has led to predictions about catalytic mechanism and design of metal-binding sites, demonstrating the strength of this interdisciplinary approach for understanding antibody structure and catalysis.

Relevant publications

Thayer, M. M., Olender, E. H., Arvai, A. S., Koike, C. K., Canestrelli, I. L., Stewart, J. D., Benkovic, S. J., Getzoff, E. D., and Roberts, V. A., (1999) "Structural Basis for Amide Hydrolysis Catalyzed by the 43C9 Antibody", J. Mol. Biol., 291, 329--345.
Crowder, M. W., Stewart, J. D., Roberts, V. A., Bender, C. J., Tevelrakh, E., Peisach, J., Getzoff, E. D., Gaffney, B. J., and Benkovic, S. J., (1995) "Spectroscopic Studies on the Designed Metal-Binding Sites of the 43C9 Single Chain Antibody", J. Amer. Chem. Soc., 117, 5627-5634.
Roberts, V. A. and Getzoff, E. D., (1995) "Metalloantibody Design", FASEB J., 9, 94-100.
Stewart, J. D., Roberts, V. A., Crowder, M. W., Getzoff, E. D., and Benkovic, S. J., (1994) "Creation of a Novel Biosensor for Zn(II)", J. Amer. Chem. Soc., 116, 415-416.
Roberts, V. A., Stewart, J. D., Benkovic, S. J., and Getzoff, E. D., (1994) "Catalytic Antibody Model and Mutagenesis Implicate Arginine in Transition-State Stabilization", J. Mol. Biol., 235, 1098-1116.
Stewart, J. D., Roberts, V. A., Thomas, N. R., Getzoff, E. D., and Benkovic, S. J., (1994) "Site-Directed Mutagenesis of Catalytic Antibody: An Arginine and a Histidine Residue Play Key Roles", Biochemistry, 33, 1994-2003.