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 mutagene sis. Hydrolysis of vasoactive intestinal polypeptide (VIP), the immuno gen employed to elicit the antibody Light chain, was reduced by >95% b y replacement of Ser27a or His93 by Ala residues. Similar reductions i n the activity were observed using synthetic protease substrates conta ining 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 w as 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 an d His93. In contrast, a Ser26 single mutant and a His27d/Asp28 double mutant displayed increased K-m, (by about tenfold) and increased turno ver (by about tenfold) using VIP as substrate. The kinetic constants f or these mutants and the wild type protein were essentially identical with Boc-Glu-Ala-Arg as substrate. Thus, two types of residues partici pating in catalysis by the light chain have been identified. Ser27a an d His93 are essential for catalysis but not for initial high affinity complexation and substrate. Ser26 and His27d or Asp28 participate in V IP binding and limit turnover indirectly. (C) 1995 Academic Press Limi ted