Pyridoxal 5′-Phosphate-dependent Catalytic Antibody

Cofactors might efficiently extend the catalytic potential of antibodies. Monoclonal antibodies against Nα-phosphopyridoxyl-L-lysine were screened for: 1) binding of 5′-phosphopyridoxyl amino acids, 2) binding of Schiff base of pyridoxal 5′-phosphate (PLP) and amino acids, the first intermediate of all PLP-dependent reactions, and 3) catalysis of PLP-dependent α,β-elimination with β-chloro-D/L-alanine. All three criteria were met by antibody 15A9. Further analysis for PLP-dependent reactions showed that this antibody catalyzes the cofactor-dependent transamination of hydrophobic D-amino acids and oxo acids (kcat′ = 0.42 min−1 with D-alanine). No other reactions with either D- or L-amino acids were taking place. PLP markedly contributes to catalytic efficacy, being a 104 times more efficient acceptor of the amino group than pyruvate. The antibody further accelerates the reaction (kcat(antibody)′/kcat(PLP)′ = 5 × 103 with D-alanine as substrate) and ensures reaction specificity, stereospecificity, as well as limited substrate specificity.