Pyridoxal-5′-phosphate-dependent catalytic antibodies

Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5′-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapt...

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Veröffentlicht in:Journal of immunological methods 2002-11, Vol.269 (1), p.99-110
Hauptverfasser: Gramatikova, Svetlana, Mouratou, Barbara, Stetefeld, Jörg, Mehta, Perdeep K, Christen, Philipp
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Sprache:eng
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Zusammenfassung:Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5′-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten N α-(5′-phosphopyridoxyl)- l-lysine, a stable analog of the covalent coenzyme–substrate adducts were screened by a competition ELISA for binding of the PLP–amino acid Schiff base adduct. The Schiff base with its C4′–Nα double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent α,β-elimination reaction of β-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic d-amino acids and oxo acids ( k cat′=0.42 min −1 with d-alanine at 25 °C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme–amino acid adduct. (2) The bond at Cα to be broken lies together with the CαN bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The α-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme–substrate adduct assumes a cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10 4 times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9 k cat (Ab·PLP)′/ k cat (PLP)′=5×10 3). The analogies of antibody 15A9 with PLP-dependent enzymes suggest that the selection criteria in the screening protocol were similar to those that have been operative in the molecular evolution of enzyme-assisted pyridoxal catalysis.
ISSN:0022-1759
1872-7905
DOI:10.1016/S0022-1759(02)00227-2