MONOCLONAL-ANTIBODIES AGAINST N-ALPHA-(5'-PHOSPHOPYRIDOXYL)-L-LYSINE - SCREENING AND SPECTRUM OF PYRIDOXAL 5'-PHOSPHATE-DEPENDENT ACTIVITIES TOWARD AMINO-ACIDS

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 generat ion of cofactor-dependent abzymes. Here we report an efficient procedu re to screen antibodies for PLP dependent catalytic activity and detai l the spectrum of catalytic activities found in monoclonal antibodies elicited against N-alpha-(5'-phosphopyridoxyl)-L-lysine. This hapten i s a nonplanar analog of the planar, resonance-stabilized coenzyme subs trate adducts formed in the PLP-dependent reactions of amino acids. Th e hapten-binding antibodies were screened for binding of the planar Sc hiff 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 mos t of the total 24 hapten-binding antibodies. Three positive clones bou nd the Schiff base with L-norleucine, two preferred that with the D-en antiomer. The positive clones were assayed for catalysis of Schiff bas e formation and of the alpha,beta-elimination reaction with the D and L-enantiomers of beta-chloroalanine. Three antibodies were found to ac celerate aldimine formation, and two of these catalyzed the PLP-depend ent alpha,beta-elimination reaction. One of the alpha,beta-elimination -positive antibodies catalyzed the transamination reaction with hydrop hobic D-amino acids and oxoacids (Gramatikova, S.I., and Christen, P. (1996) J. Biol. Chem. 271, 30583-30586). All catalytically active anti bodies displayed continuous turnover. No PLP-dependent reactions other than aldimine formation, alpha,beta-elimination of beta-chloroalanine and transamination were detected. The successive screening steps plau sibly simulate the functional selection pressures having been operativ e in the molecular evolution of primordial PLP-dependent protein catal ysts to reaction- and substrate-specific enzymes.