Crossreactivity, efficiency and catalytic specificity of an esterase-like antibody

The antibody D2.3 catalyzes the hydrolysis of several p-nitrobenzyl and p-n itrophenyl esters with significant rate enhancement; product inhibition is observed with the former compounds but not with the latter. Whereas enzyme specificity has been extensively studied by X-ray crystallography, structur al data on catalytic antibodies have thus far related only to one of the re actions they catalyze. To investigate the substrate specificity and the sub strate relative to product selectivity of D2.3, we have determined the stru ctures of its complexes with two p-nitrophenyl phosphonate transition state analogs (TSAs) and with the reaction product, p-nitrophenol. The complexes with these TSAs, determined at 1.9 Angstrom resolution, and that with p-ni trobenzyl phosphonate determined previously, differ mainly by the locations and conformations of the ligands. Taken together with kinetic data, the st ructures suggest that a hydrogen bond to an atom of the substrate distant b y eight covalent bonds from the carbonyl group of the hydrolyzed ester bond contributes to catalytic efficiency and substrate specificity. The structu re of Fab D2.3 complexed with p-nitrophenol was determined at 2.1 Angstrom resolution. Release of p-nitrophenol is facilitated due to the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate w ith the hydrophobic cavity where it is located, and to the absence of a dir ect hydrogen bond between the product and the Fab. Catalytic specificity an d the manner of product release are both affected by interactions with subs trate atoms remote from the reaction center that were not programmed in the design of the TSA used to elicit this antibody. Selection of a catalytic a ntibody that makes use of TSA unprogrammed features has been made practical because of the screening for catalytic efficiency incorporated in the proc edure used to obtain it. (C) 1998 Academic Press.