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Article Dans Une Revue Biochemical Journal Année : 2000

Mechanism of an antibody-catalysed allylic isomerization

Résumé

The catalytic antibody 4B2, which was generated against a substituted amidine 1, catalyses the allylic isomerization of β,γ-unsaturated ketones with an acceleration factor (kcat/kuncat) of 1.5×103. On the basis of the ‘bait and switch’ strategy, it was reasoned that the positively charged hapten could elicit, by charge complementarity, an acidic residue (Asp or Glu) in the antibody-binding site in the right position to catalyse this proton transfer reaction. The pH dependence curve of kcat/Km shows a bell-shaped feature with an optimum at approx. pH 4.5. By cloning and sequencing the light and heavy chains of the 4B2 antibody, we confirmed the presence of several Asp and Glu residues in the complementarity-determining region loops. The antibody catalyses the α-proton exchange on the same substrates, demonstrating the involvement of a dienol intermediate in the reaction mechanism. Kinetic studies with 2H-NMR provide evidence that α-proton abstraction is stereospecific. Whether the process involves one or two acid/base residues in this simple proton transfer or whether it is a concerted mechanism is discussed.

Domaines

Biotechnologie

Dates et versions

hal-01888046 , version 1 (04-10-2018)

Identifiants

Citer

Olivier Gonçalves, Thierry Dintinger, Jacques Lebreton, Dominique Blanchard, Charles Tellier. Mechanism of an antibody-catalysed allylic isomerization. Biochemical Journal, 2000, 346 (3), ⟨10.1042/0264-6021:3460691⟩. ⟨hal-01888046⟩
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