Site-directed mutagenesis of proteolytic antibody light chain.
Amino acid residues in a proteolytic antibody light chain selected by molecular modeling were substituted with Ala by site-directed mutagenesis. Hydrolysis of vasoactive intestinal polypeptide (VIP), the immunogen employed to elicit the antibody light chain, was reduced by > 95% by replacement of Ser27a or His93 by Ala residues. Similar reductions in the activity were observed using synthetic protease substrates containing Arg-methylcoumarinamide (MCA) and Lys-MCA bonds. Turnover of the Ser27a and His93 mutants was lower than that of wild-type protein by about two orders of magnitude. The activity of the wild-type protein was inhibited selectively by diisopropylfluorophosphate (DFP), a serine protease inhibitor, but the residual activity of the Ser26 mutant was refractory to DFP. The affinity of the wild-type light chain for the substrate ground state was nearly unaffected by mutations at Ser27a and His93. In contrast, a Ser26 single mutant and a His27d/Asp28 double mutant displayed increased Km (by about tenfold) and increased turnover (by about tenfold) using VIP as substrate. The kinetic constants for these mutants and the wild type protein were essentially identical with Boc-Glu-Ala-Arg as substrate. Thus, two types of residues participating in catalysis by the light chain have been identified. Ser27a and His93 are essential for catalysis but not for initial high affinity complexation and substrate. Ser26 and His27d or Asp28 participate in VIP binding and limit turnover indirectly.
D000595:Amino Acid Sequence; D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D001665:Binding Sites; D018076:DNA, Complementary / Q000235:genetics; D005803:Genes, Immunoglobulin; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014660:Vasoactive Intestinal Peptide / Q000276:immunology
D017151:Antibodies, Catalytic; D018076:DNA, Complementary; D007145:Immunoglobulin kappa-Chains; D015842:Serine Proteinase Inhibitors; D007531:Isoflurophate; D014660:Vasoactive Intestinal Peptide