Identification of functionally important residues in the pyridoxal-5'-phosphate-dependent catalytic antibody 15A9.
Antibody 15A9 is unique in its ability to catalyze the transamination reaction of hydrophobic D-amino acids with pyridoxal-5'-phosphate (PLP). Both previous chemical modification studies and a three dimensional (3-D) homology model indicated the presence of functionally important tyrosine residues in the antigen-binding cavity of antibody 15A9. To gain further insight into the hapten, ligand binding, and catalytic mechanism of 15A9, all tyrosine residues in the complementarity-determining regions (CDRs) and the single arginine residue in CDR3 of the light chain were subject to an alanine scan. Substitution of Tyr(H33), Tyr(L94), or Arg(L91) abolished the catalytic activity and reduced the affinity for PLP and N(a)-(5'-phosphopyridoxyl)-amino acids, which are close analogues of covalent PLP-substrate adducts. The Tyr(H100b)Ala mutant possessed no detectable catalytic activity, while its affinity for each ligand was essentially the same as that of the wild-type antibody. The binding affinity for the hapten was drastically reduced by a Tyr(L32)Ala mutation, suggesting that the hydroxyphenyl group of Tyr(L32) participates in the binding of the extended side chain of the hapten. The other Tyr --> Ala substitutions affected both binding and catalytic activity only to a minor degree. On the basis of the information obtained from the mutagenesis study, we docked N(alpha)-(5'-phosphopyridoxyl)-D-alanine into the antigen-binding site. According to this model, Arg(L91) binds the alpha-carboxylate group of the amino acid substrate and Tyr(H100b) plays an essential role in the catalytic mechanism of antibody 15A9 by facilitating the Calpha/C4' prototropic shift. In addition, the catalytic apparatus of antibody 15A9 revealed several mechanistic features that overlap with those of PLP-dependent enzymes.
Mouratou
Barbara
B
Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. mouratou@lebs.cnrs-gif.fr
Stetefeld
Jörg
J
D000409:Alanine; D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001666:Binding Sites, Antibody; D006241:Haptens; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D017151:Antibodies, Catalytic; D006241:Haptens; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D000409:Alanine