Abstract
Cofactors might efficiently extend the catalytic potential of antibodies. Monoclonal antibodies against Nalpha-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 alpha,beta-elimination with beta-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 10(4) times more efficient acceptor of the amino group than pyruvate. The antibody further accelerates the reaction (kcat(antibody)'/kcat(PLP)' = 5 x 10(3) with D-alanine as substrate) and ensures reaction specificity, stereospecificity, as well as limited substrate specificity.
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