MECHANISTICALLY DIFFERENT CATALYTIC ANTIBODIES OBTAINED FROM IMMUNIZATION WITH A SINGLE TRANSITION-STATE ANALOG

The variable-region peptide sequence and steady-state kinetic behavior are compared for a family of catalytic antibodies that arose from the same immune response to a transition-state analog, The crystal struct ure of the most catalytically active member of the family (17E8) has b een solved to 2.5 Angstrom resolution and shows that the antibody acti ve site contains a Ser(H99)-His(H35) (H = heavy chain) catalytic dyad analogous to the Ser-His-Asp catalytic triad of serine proteases, The variable-region peptide sequence of the next most active antibody (29G 11) differs from that of 17E8 by nine heavy-chain point mutations, and results from computer modeling suggest that the three-dimensional str ucture of 29G11 is similar to that of 17E8. In addition, 29G11 is an e fficient catalytic antibody; it possesses 26% of the hydrolytic activi ty of 17E8. There is one active-site mutation in 29G11 compared to 17E 8; position 99 of the heavy chain of 29G11 contains a glycine residue in place of the nucleophilic serine at this position in 17E8. Consiste nt with this mutation, results from pH-rate studies and hydroxylamine partitioning experiments indicate that in contrast to the catalytic me chanism of 17E8, the mechanism of 29G11-catalyzed esterolysis does not feature nucleophilic catalysis.