Amino acid sequences and hapten binding of catalytic and noncatalytic antibodies against Nα-(5′-phosphopyridoxyl)- l-lysine

Abstract

Upon immunization with a transition-state analog, only a minority of the hapten-binding antibodies will possess catalytic activity, which will vary in efficacy and substrate specificity. Here, the amino acid sequences of the variable domains of two pyridoxal-5′-phosphate-dependent catalytic and five noncatalytic hapten-binding antibodies raised by immunization with protein-conjugated N α-(5′-phosphopyridoxyl)- l-lysine (Gramatikova, S., Christen, P., 1997. J. Biol. Chem. 272, 9779–9784) were determined by sequencing their cDNAs. The analysis revealed that the light chains of this set of antibodies were closely related (pairwise identity 65–80%), whereas the heavy chains could be traced back to two different but related groups (intergroup identity 50–54%). The majority of the antibodies proved not to be clonally related, a finding which correlates with their differences in enantiomeric selectivity in ligand binding and reaction specificity. Only one noncatalytic antibody was found to be clonally related with a catalytic antibody, the sequence identity being >95% in both the V H and V L domains. The complementarity-determining regions were invariably abundant in tyrosine residues. Nitration of three to four tyrosine residues with tetranitromethane abolished hapten binding and catalytic activity. Partial protection by pyridoxal-5′-phosphate against inactivation suggested the presence of functionally important tyrosine residues in the binding sites of the antibodies.