Monoclonal Antibodies against Nα-(5′-Phosphopyridoxyl)-L-lysine

Cofactors may be expected to expand the range of reactions amenable to antibody-assisted catalysis. The biological importance of pyridoxal 5′-phosphate (PLP) as enzymic cofactor in amino acid metabolism and its catalytic versatility make it an attractive candidate for the generation of cofactor-dependent abzymes. Here we report an efficient procedure to screen antibodies for PLP-dependent catalytic activity and detail the spectrum of catalytic activities found in monoclonal antibodies elicited against N α -(5′-phosphopyridoxyl)- L -lysine. This hapten is a nonplanar analog of the planar, resonance-stabilized coenzyme-substrate adducts formed in the PLP-dependent reactions of amino acids. The hapten-binding antibodies were screened for binding of the planar Schiff base formed from PLP and D - or L -norleucine by competition enzyme-linked immunosorbent assay. The Schiff base (external aldimine) is an obligatory intermediate in all PLP-dependent reactions of amino acids. This simple, yet highly discriminating screening step eliminated most of the total 24 hapten-binding antibodies. Three positive clones bound the Schiff base with L -norleucine, two preferred that with the D -enantiomer. The positive clones were assayed for catalysis of Schiff base formation and of the α,β-elimination reaction with the D - and L -enantiomers of β-chloroalanine. Three antibodies were found to accelerate aldimine formation, and two of these catalyzed the PLP-dependent α,β-elimination reaction. One of the α,β-elimination-positive antibodies catalyzed the transamination reaction with hydrophobic D -amino acids and oxoacids (Gramatikova, S. I., and Christen, P. (1996) J. Biol. Chem. 271, 30583-30586). All catalytically active antibodies displayed continuous turnover. No PLP-dependent reactions other than aldimine formation, α,β-elimination of β-chloroalanine and transamination were detected. The successive screening steps plausibly simulate the functional selection pressures having been operative in the molecular evolution of primordial PLP-dependent protein catalysts to reaction- and substrate-specific enzymes.