Amino acid sequences and hapten binding of catalytic and noncatalytic antibodies against N-alpha-(5 '-phosphopyridoxyl)-L-lysine

Upon immunization with a transition-state analog, only a minority of the ha pten-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-alpha-(5'-phosphopyridoxyl)-L-lysine (Gramatikova, S., Christ en, 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 ant ibodies 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 noncataly tic antibody was found to be clonally related with a catalytic antibody, th e sequence identity being > 95% in both the V-H and V-L domains. The comple mentarity-determining regions were invariably abundant in tyrosine residues . Nitration of three to four tyrosine residues with tetranitromethane aboli shed hapten binding and catalytic activity. Partial protection by pyridoxal -5'-phosphate against inactivation suggested the presence of functionally i mportant tyrosine residues in the binding sites of the antibodies. (C) 2001 Elsevier Science Ltd. All rights reserved.