Structural studies of catalytic antibody mechanisms

Catalytic antibody 21H3 catalyzes the bimolecular transesterification of an ester substrate with a second alcohol substrate with a stereocenter. Unexpectedly, it was observed that the transesterification reaction follows a ping-pong mechanism not programmed in the antibody hapten. The ping-pong mechanism involves a two-step reaction, in which the first substrate reacts with the antibody and produces a covalent intermediate. This first reaction is followed by the entrance of the second substrate into the antibody active site, which reacts with the covalent intermediate to form the final product. Crystal structures of antibody 21H3 unliganded and bound with (S)-hapten, were solved to resolutions of 2.0A and 1.8A, respectively. Based on the crystal structures, Ser 100 (H) in the CDR H3 loop was proposed to be the attachment site of the covalent intermediate. A relatively sophisticated mechanism is proposed for 21H3 transesterification, in which activation of the reactive serine alcohol depends on ligand binding.; In addition to having an unusual mechanism, antibody 21H3 also possesses a paradoxical stereoselectivity for its second substrate. The antibody selectively catalyzes the (S)-enantiomer of the second substrate and does not react with the (R)-enantiomer.; The structural mechanism of a photoactive catalytic antibody, 19G2, was also studied. Antibody 19G2 was part of a panel of antibodies raised against a trans-stilbene hapten. By itself, stilbene emits a relatively weak violet fluorescence. However, when stilbene is mixed with some of the anti-stilbene antibodies, such as 1962, it unexpectedly emits an intense blue fluorescence with a quantum yield of nearly 0.8.; In addition to its putative exciplex mechanism, the fluorescence showed an unusual temperature dependence. Above 260K, the stilbene-antibody complex emitted its characteristic blue fluorescence, at 240K and below, the blue fluorescence disappeared, leaving only the weak violet fluorescence of non-exciplex stilbene. The crystal structure of 19G2 in complex with the stilbene hapten at 277K indicated that the antibody and stilbene atoms were in virtually the same positions at both temperatures. Thus the temperature-dependent loss of the blue fluorescence is likely to be due to the quenching of transient motion not directly observed in the crystal structures. (Abstract shortened by UMI.)...