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.