STRUCTURES, MECHANISM AND SPECIFICITY OF A CATALYTIC ANTIBODY

Benoît Gigant¹, Jean-Baptiste Charbonnier¹, Zelig Eshhar², Bernard S. Green³, and Marcel Knossow¹.

¹Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Bat. 34, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France;

²Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel and
³Department of Pharmaceutical Chemistry, The Hebrew University School of Pharmacy, Jerusalem 91120, Israel.

E-mail: gigant@lebs.cnrs-gif.fr

We report the study of an antibody, elicited by immunization with a p-nitrobenzyl phosphonate, that catalyses the hydrolysis of the corresponding p-nitrobenzyl ester. We have determined the X-ray structure of the Fab complexed with a substrate analogue amide, with the phosphonate transition state analogue (TSA) and with the alcohol product of the reaction. The deduced reaction pathway follows. The antibody acts by preferential stabilisation of the negatively charged oxyanion intermediate of the reaction that results from hydroxide attack on the substrate. A tyrosine residue plays a crucial role in catalysis: it activates the ester substrate and, together with an asparagine, it stabilises the oxyanion intermediate. A canal allows facile diffusion of water molecules to the reaction centre, which is deeply buried in the structure.

In addition to esters of p-nitrobenzyl alcohol, the antibody catalyses the hydrolysis of p-nitrophenyl esters. While the crystal structures of the Fab complexed with two different p-nitrophenyl phosphonates suggest a similar catalytic mechanism, these new structures shed light on two features of this antibody that govern its substrate specificity. First, lesser ground state stabilisation accounts for the larger rate enhancement of the hydrolysis of p-nitrobenzyl esters compared to p-nitrophenyl esters; second, a hydrogen bond to an atom of the substrate distant by eight covalent bonds from the carbonyl of the hydrolysed ester bond contributes to catalytic efficiency and substrate specificity. Interestingly, unlike p-nitrobenzyl alcohol, p-nitrophenol does not inhibit the reaction. Release of p-nitrophenol is facilitated by the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate with the hydrophobic cavity where it is located and by the absence of a direct hydrogen bond between the product and the Fab. When product release is taken into account, the antibody catalyses the hydrolysis of p-nitrophenyl esters more efficiently than that of the initially designed target subsrate, p-nitrobenzyl esters.