title: Characterization of the mechanism of action of a catalytic antibody.
abstract: The time course of phenylacetate hydrolysis by the catalytic antibody 20G9 has a kinetic burst lasting several reaction cycles. The burst is caused by partial mixed inhibition by one product of the hydrolysis, phenol, which binds with an apparent dissociation constant of 4.6 microM. Phenol binding causes kcat to decrease from 9.1 min-1 to 1.0 min-1 and Km to decrease from 300 microM to 36 microM. Because Km decreases but kcat/Km is unaffected, phenol must perturb the ground state structure but not the transition state structure. Structural complementarity to the transition state seems to be an important contributor to catalysis by 20G9 because weak binding in the ground state can be markedly improved by adding phenol, but tight binding of the transition state, which has been optimized by the immune system, cannot be readily improved. Further evidence that the substrate ground and transition states differ greatly in complementarity to the antibody is that the substrate binds more than five orders of magnitude more weakly than the transition state analogue hapten to which the antibody was raised. Two additional phenol molecules bind at higher product concentrations; the first binds over the concentration range of 15 to 86 microM and accelerates hydrolytic activity by 42%; the second is a competitive inhibitor with a Ki of 140 microM. Binding of multiple phenol molecules suggests the presence of abundant hydrophobic amino acids in the complementarity-determining region of 20G9.
authors.affiliation: IGEN Inc, Rockville, MD 20852.
authors.forename: M T
authors.initials: MT
authors.lastname: Martin
authors.forename: A R
authors.initials: AR
authors.lastname: Schantz
authors.forename: P G
authors.initials: PG
authors.lastname: Schultz
authors.forename: A R
authors.initials: AR
authors.lastname: Rees
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D002384:Catalysis; D008956:Models, Chemical; D015394:Molecular Structure; D019800:Phenol; D010636:Phenols
chemical_list: D000906:Antibodies; D010636:Phenols; D019800:Phenol